Note: Descriptions are shown in the official language in which they were submitted.
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Antimicrobial Compositions and Formulations
This invention relates to antimicrobial compositions and formulations,
particularly colloids,
suspensions or emulsions. The compositions and formulations may be for topical
application for the treatment of antimicrobial infections, such as viral,
bacterial, or fungal
infections.
Cold sores are small blisters that develop on the lips or around the mouth.
They are caused
by the herpes simplex virus (HSV) and usually clear up without treatment
within 7 to 10
days. Cold sores often start with a tingling, itching or burning sensation
around the mouth.
Small fluid-filled sores will then appear, most commonly on the edges of the
lower lip.
Topical products, such as creams or gels, can be used to treat cold sores.
Many are
prescription medicines that may slightly shorten the duration of cold sores,
usually by just 1
to 2 days.
Genital herpes is a common infection caused by the herpes simplex virus (HSV).
It causes
painful blisters on the genitals and the surrounding areas. Herpes can be
treated with anti-
viral agents. However, these can cause side effects, such as nausea and
headaches.
There remains a need for topical formulations that provide effective treatment
of HSV and
other viral or microbial infections.
The Applicant has found that compositions that are able to release hydrogen
peroxide at
the site of a microbial infection are particularly effective at preventing or
inhibiting the
infection.
SurgihoneyTM is a chemical engineered honey that has the ability to deliver
variable and
sustained doses of reactive oxygen species (ROS). Studies in vitro and in vivo
have
demonstrated Surgihoney's efficacy in eradication of infection. This has
included drug
resistant strains, such as methicillin-resistant S. aureus (MRSA) and
vanomycin-resistant
Enterococcus faecium (Dryden, M., Lockyer, G., Saeed, K., & Cooke, J. (2014).
Engineered Honey: In Vitro Antimicrobial Activity of a Novel Topical Wound
Care
Treatment. Journal of Global Antimicrobial Resistance, 2, 168-172). It was
also shown to
be effective against fungi and prevented or reduced the seeding of biofilms
(Dryden, M.,
Halstead, F., & Cooke, J. (2015). Engineered Honey to Manage Bacterial
Bioburden and
Biofilm in Chronic Wounds. EWMA Free Paper Session: Infection and
Antimicrobials).
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Chemically engineered honeys, such as honey with added glucose oxidase, are
disclosed
in WO 2015/166197 Al.
At present, SurgihoneyTM is available in a sachet form for topical
administration. However,
administration of SurgihoneyTM in this form may be inconvenient, make
application of a
controlled dose difficult and may be clinically non-optimal.
According to the invention there is provided a composition for generating
antimicrobial
activity, which comprises: a lipophilic phase; an aqueous phase; an enzyme
that is able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a
substrate for the enzyme. A composition of the invention may be in the form of
a colloid or
a suspension.
The term "colloid" is used herein to refer to a homogeneous non-crystalline
substance
consisting of large molecules or ultramicroscopic particles of one substance
dispersed
through a second substance. Colloids include gels, sols, and emulsions. The
particles do
not settle, and cannot be separated out by ordinary filtering or centrifuging
like those in a
suspension.
The term "suspension" is used herein to refer to a mixture in which small
particles of a
substance are dispersed throughout a liquid. If a suspension is left
undisturbed, the
particles are likely to settle to the bottom. The particles in a suspension
are larger than
those in either a colloid or a solution.
A composition of the invention may be in the form of an emulsion. The term
"emulsion" is
used herein to refer to a fine dispersion of minute droplets of one liquid in
another in which
it is not soluble or miscible. An emulsion of the invention may be an oil and
water emulsion,
in particular an oil-in-water emulsion, or a water-in-oil emulsion. The
composition may be a
micro-emulsion.
Compositions of the invention may comprise a first phase (or first liquid, or
first component)
and a second phase (or second liquid, or second component), an enzyme that is
able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a
substrate for the enzyme. The first phase and the second phase may be
immiscible. For
example, the first phase may be less polar than the second phase. The first
phase may be
a non-polar phase such as a lipophilic phase or a hydrophobic phase e.g. an
oil. The
second phase may be a polar phase, such as an aqueous phase. The second phase
may
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comprise a non-aqueous solvent. Droplets or micelles of the second phase may
be
dispersed within the first phase.
The second phase may comprise water and/or non-aqueous solvent. The enzyme and
the
substance of the composition may be dissolved in the water and/or non-aqueous
solvent.
It is conceivable that, in some embodiments, the second phase may not comprise
water or
may comprise substantially no water. In such circumstances, the second phase
may be
described as non-aqueous. For example, the enzyme and the substance comprising
a
substrate for the enzyme may be dissolved in a non-aqueous solvent. The non-
aqueous
solvent may be immiscible with respect to the first phase e.g. lipophilic
phase.
In some embodiments, the enzyme that is able to convert a substrate to release
hydrogen
peroxide and the substance that includes a substrate for the enzyme may be
contained
within micelles dispersed within the first phase, e.g. lipophilic phase.
In some compositions, the composition may be in the form of a double emulsion.
For
example, droplets containing the enzyme that is able to convert a substrate to
release
hydrogen peroxide and the substance that includes a substrate for the enzyme
may be
dispersed within globules of a lipophilic phase (e.g. oil globules) and
globules may be
dispersed within an aqueous phase. Such a double emulsion may be termed a
water-in-oil-
in-water type (W/O/W) emulsion.
A composition of the invention may further comprise an emulsifying agent (or
emulsifier).
Emulsions can be stabilized by adsorption of surface active agents
(emulsifying agents) at
the emulsion interface. Emulsifying agents lower the interfacial tension to
maintain the
droplets in a dispersed state. An emulsifying agent has a hydrophilic part and
a lipophilic
part. It is possible to calculate the relative quantities of an emulsifying
agent(s) necessary
to produce the most physically stable emulsions for a particular formulation
with water
combination. This approach is called the hydrophilic-lipophilic balance (HLB)
method ("The
HLB SYSTEM a time-saving guide to emulsifier selection" ICI Americas Inc.,
Wilmington,
Delaware 19897, 1976, revised 1980). Each emulsifying agent is allocated an
HLB number
representing the relative properties of the lipophilic and hydrophilic parts
of the molecule.
High numbers (up to a theoretical number of 20), indicates an emulsifying
agent exhibiting
mainly hydrophilic or polar properties, whereas low numbers represent
lipophilic or non-
polar characteristics. According to the HLB System, all fats and oils have a
Required HLB.
Emulsions with optimal performance can be yielded by matching the HLB
requirement with
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the emulsifying agent's HLB value. For an oil-in-water emulsion, the more
polar the oil
phase the more polar the emulsifying agent(s) must be. For example, to
emulsify Soybean
Oil, which has a Required HLB of 7, according to the HLB system, it would be
necessary to
use an emulsifying agent, or blend of emulsifying agents, with an HLB of 7
1. The HLB of
emulsifying agents can be calculated or determined through trial and error.
Thus, the lipophilic phase of a composition of the invention may require an
emulsifying
agent of a particular HLB number in order to ensure a stable product. The
lipophilic phase
of a composition of the invention may comprise an oil or a wax. Examples of
oils and
waxes (by their International Nomenclature of Cosmetic Ingredients, INCI,
name) for use in
a lipophilic phase of a composition of the invention (with their respective
Required HLBs)
include the following:
Aleurites Moluccana Seed Oil [7] Grape (Vitis Vinifera) Seed Oil [7]
Almond Oil NF [6] Hybrid Safflower (Carthamus
Tinctorius) Oil [9]
Anhydrous Lanolin USP [10] Isopropyl Myristate [11.5]
Apricot Kernel Oil [7] Isopropyl PaImitate [11.5]
Avocado (Persea Gratissima) Oil [7] Jojoba (Buxus Chinensis) Oil [6.5]
Babassu Oil [8] Lanolin [10]
Beeswax [12] Macadamia (Ternifolia) Nut Oil [7]
Borage (Borago Officinalis) Seed Oil [7] Mangifera Indica (Mango) Seed
Butter [8]
Brazil Nut Oil [8] Mineral Oil [10.5]
C12-15 Alkyl Benzoate [13] Myristyl Myristate [8.5]
Cannabis Sativa Seed Oil [7] Olive (Olea Europaea) Oil [7]
Canola Oil [7] Oryza Sativa (Rice Bran) Oil [7]
Caprylic/Capric Triglyceride [5] Peanut Oil NF [6]
Carrot (Daucus Carota Sativa) Seed Oil [6] Petrolatum [7]
Castor (Ricinus Communis) Oil [14] PPG-15 Stearyl Ether [7]
Ceresin [8] Retinyl PaImitate [6]
Cetearyl Alcohol [15.5] Safflower (Carthamus Tinctorius) Oil
[8]
Cetyl Alcohol [155] Sesame (Sesamum lndicum) Oil [7]
Cetyl Esters [10] Shea Butter (Butyrospermum Parkii) [8]
Cetyl PaImitate [10] Soybean (Glycine Soja) Oil [7]
Coconut Oil [8] Stearic Acid [15]
Daucus Carota Sativa (Carrot) Root Stearyl Alcohol [15.5]
Extract [6]
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Diisopropyl Adipate [9] Sunflower (Helianthus Annus) Oil [7]
Dimethicone [5] Sweet Almond (Prunus Amygdalus
Dulcis) Oil [7]
Dog Rose (Rosa Canina) Hips Oil [7] Theobroma Cacao (Cocoa) Seed Butter
[6]
5 Emu Oil [8] Tocopherol [6]
Evening Primrose Oil [7]
In some embodiments, the lipophilic phase of a composition of the invention
comprises a
beeswax.
In some embodiments, the lipophilic phase is an oil. In some embodiments, the
oil is
selected from olive oil, corn oil, vegetable oil, sunflower oil or paraffin
oil. In a preferred
embodiment, the oil may be olive oil. In another preferred embodiment, the oil
may be
paraffin oil.
Water-in-oil emulsifying agents for use in compositions of the invention may
have an HLB
value in the range 3-6. Oil-in-water emulsifying agents for use in
compositions of the
invention may have an HLB value in the range 8-18. Examples of emulsifying
agents (by
their INCI name) for use in compositions of the invention (with their HLB
numbers) include
the following:
Calcium Stearoyl Lactylate [HLB = 5.1 1] Oleth-20 [HLB = 15.3 1]
Ceteareth-20 [HLB = 15.2 1] PEG-100 Stearate [HLB = 18.8 1]
Cetearyl Glucoside [HLB = 11 1] PEG-20 Almond Glycerides [HLB = 10 1]
Ceteth-10 [HLB = 12.9 1] PEG-20 Methyl Glucose Sesquistearate
[HLB = 15 1]
Ceteth-2 [HLB = 5.3 1] PEG-25 Hydrogenated Castor Oil
[HLB = 10.8 1]
Ceteth-20 [HLB = 15.7 1] PEG-30 Dipolyhydroxystearate [HLB = 5.5 1]
Cocamide MEA [HLB = 13.5 1] PEG-4 Dilaurate [HLB = 6 1]
Glyceryl Laurate [HLB = 5.2 1] PEG-40 Sorbitan Peroleate [HLB = 9
1]
Glyceryl Stearate [HLB = 3.8 1] PEG-60 Almond Glycerides [HLB = 15
1]
Glyceryl Stearate (and) PEG-100 Stearate PEG-8 Laurate [HLB = 13 1]
[HLB = 11 1]
Glyceryl Stearate SE [HLB = 5.8 1] PEG-80 Sorbitan Laurate [HLB = 19.1
1]
Glycol Distearate [HLB = 1 1] Polysorbate 20 [HLB = 16.7 1]
Glycol Stearate [HLB = 2.9 1] Polysorbate 60 [HLB = 14.9 1]
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isoceteth-20 [HLB = 15.7 1] Polysorbate 80 [HLB = 15 1]
lsosteareth-20 [HLB = 15 1] Polysorbate 85 [HLB = 11 1]
Lauramide DEA [HLB = 15 1] Sodium Stearoyl Lactylate [HLB = 8.3
1]
Laureth-23 [HLB = 16.9 1] Sorbitan lsostearate [HLB = 4.7 1]
Laureth-4 [HLB = 9.7 1] Sorbitan Laurate [HLB = 8.6 1]
Lecithin [HLB = 4 1] Sorbitan Oleate [HLB = 4.3 1]
Lecithin [HLB = 9.7 1] Sorbitan Sesquioleate [HLB = 3.7 1]
Linoleamide DEA [HLB = 10 1] Sorbitan Stearate [HLB = 4.7 1]
Methyl Glucose Sesquistearate Sorbitan Stearate (and) Sucrose
Cocoate
[HLB = 6.6 1] [HLB = 6 1]
Oleth-10 [HLB = 12.4 1] Sorbitan Trioleate [HLB = 1.8 1]
Oleth-10 / Polyoxyl 10 Oleyl Ether NF Stearamide MEA [HLB = 11 1]
[HLB = 12.4 1]
Oleth-2 [HLB = 4.9 1] Steareth-2 [HLB = 4.9 1]
Oleth-20 [HLB = 12.4 1] Steareth-21 [HLB = 15.5 1]
In some embodiments, an emulsifying agent of a composition of the invention
comprises a
lecithin.
Emulsifying agents include ionic or non-ionic surfactants, and lipophilic
fatty amphiles (for
example, fatty alcohols or fatty acids). Non-ionic surfactants may be
preferred since they
may be less irritating to skin that anionic or cationic surfactants.
Other examples of suitable emulsifying agents include: Surfactants: Sodium
lauryl
sulphate, Cetrimide, Cetomacrogol 1000, PEG 1000 monostearate, Triethanolamine
stearate, Sodium stearate; Fatty amphiphiles: Cetostearyl alcohol, Cetyl
alcohol, Stearyl
alcohol, Glyceryl monostearate, Stearic acid, Phosphatidylcholine.
Examples of commercial emulsifying waxes include: Emulsifying wax BP
(Cetostearyl
alcohol, sodium lauryl sulphate), Emulsifying wax USNF (Cetyl alcohol,
polysorbate),
Cationic emulsifying wax BPC (Cetostearyl alcohol, cetrimide), Glyceryl
monostearate S.E.
(Glyceryl monostearate, sodium stearate), Cetomacrogol emulsifying wax BPC
(Cetostearyl alcohol, cetomacrogol 1000), Polawax (Cetyl alcohol, non-ionic
surfactant),
Lecithin (Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol,
phosphatidic acid).
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Surfactants for use in compositions of the invention may include one or more
of TVVEEN
(e.g. TVVEEN 80), SPAN (e.g. SPAN 80), Poloxamer (e.g. Poloxamer 407) and
Polyglycerol polyricinoleate (PGPR). A preferred surfactant may be Poloxamer,
such as
Poloxamer 407. Another preferred surfactant may be PGPR.
Surfactants may include a surfactant polymer, or co-polymer. For example, a
suitable
surfactant may be a triblock copolymer consisting of a central hydrophobic
block flanked by
two hydrophilic blocks.
According to the invention, there is provided a composition for generating
antimicrobial
activity, which comprises: an oil; an emulsifier; an enzyme that is able to
convert a
substrate to release hydrogen peroxide; and a substance that includes a
substrate for the
enzyme.
Compositions of the invention may comprise non-aqueous solvent. The non-
aqueous
solvent may be a polar solvent, such as a solvent with a dielectric constant
of greater than
15. The non-aqueous solvent may be an organic solvent. For example, the
solvent may be,
or may comprise, glycerol, dimethylsulphoxide, propylene glycol or
polyethylene glycol. The
non-aqueous solvent may be immiscible with respect to the first phase e.g. the
lipophilic
phase (such as oil).
In a preferred embodiment, the non-aqueous solvent may be, or comprise,
glycerol.
In some embodiments, compositions of the invention may comprise micelles,
preferably
reverse micelles. Within each micelle may be the enzyme and the substance
(which may
comprise an unrefined natural substance, such as honey), and outside of the
micelle may
be the first phase, e.g. the lipophilic phase (such as oil). Within each
reverse micelle there
may also be water and/or non-aqueous solvent. Within each micelle there may
not be
sufficient water for the enzyme to convert the substrate.
Compositions of the invention may comprise further components which may assist
in
reducing coalescence. Coalescence describes the situation in which two or more
droplets,
or micelles, combine to form a single droplet, or micelle. In order to reduce
or prevent,
coalescence, the strength of the interfacial film, i.e. the interface between
the lipophilic
phase and the aqueous phase, may be strengthened. This may be achieved, for
example,
by increasing the surfactant concentration, including an anwhiphilic polymer,
and/or by
adding an alcohol, such as an aliphatic alcohol with 5-7 carbon atoms.
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Compositions of the invention may be suitable for topical application, in
particular topical
application to a human subject. A composition for topical application may be
applied to
body surfaces such as skin or mucous membranes. Compositions of the invention
for
topical application may be in the form, for example, of a cream, a lotion, or
a lip balm.
The term "cream" is used herein to refer to a semi-solid emulsion of oil-in-
water, or water-
in-oil, for topical use. Oil-in-water (o/w) creams are composed of small
droplets of oil
dispersed in a continuous aqueous phase, and water-in-oil (w/o) creams are
composed of
small droplets of water dispersed in a continuous oily phase. Oil-in-water
creams are less
greasy and more easily washed off using water. Water-in-oil creams are more
moisturising
as they provide an oily barrier which reduces water loss from the outermost
layer of the
skin.
The term "cream" may also refer to a semi-solid emulsion in which droplets of
a first phase
are dispersed in a continuous second phase, or in which droplets of a second
phase are
dispersed in a continuous first phase. For example, the first phase may be
less polar than
the second phase. The first phase may be a non-polar phase such as a
lipophilic phase or
a hydrophobic phase e.g. an oil. The second phase may be a polar phase, such
as an
aqueous phase. The second phase may comprise a non-aqueous solvent. The second
phase may comprise water and/or non-aqueous solvent. It is conceivable that,
in some
embodiments, the second phase may not comprise water or may comprise
substantially no
water. In such circumstances, the second phase may be described as non-
aqueous. The
non-aqueous solvent may be immiscible with respect to the first phase e.g.
lipophilic phase.
The term "lotion" is used herein to refer to a liquid suspension or emulsion
for topical
application. A lotion may comprise finely powdered, insoluble solids held in
suspension by
suspending agents and/or surface-active agents, or an emulsion (particularly,
an oil-in-
water emulsion) stabilized by one or more surface-active agents. A lotion has
lower
viscosity than a cream.
The term "lip balm" is used herein the refer to a wax-like substance applied
topically to the
lips of the mouth to moisturize and relieve chapped or dry lips. Lip balm may
include, for
example, beeswax or carnauba wax, camphor, cetyl alcohol, lanolin, paraffin,
and
petrolatum, among other ingredients.
Advantageously, compositions of the invention may be sprayable. For example,
this may
assist in overcoming some of the difficulties in applying Surgihoney in its
conventional form.
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For example, in some circumstances, Surgihoney (which may be sticky and
viscous) may
be difficult to apply to an infection site. Consequently, compositions of the
invention may be
delivered to a patient using a spray device. The device may be a spraying or
atomising
device, such as a pump-action spray or an aerosol spray. The invention may
thus provide a
spraying device comprising a composition of the invention.
Compositions of the invention may be suitable for internal administration to a
subject. For
example, the composition may be suitable for administration into a subject's
respiratory
tract. Surgihoney, in its conventional form, may not be easily administered to
a subject's
respiratory tract,
Compositions of the invention may be administered to the respiratory tract
using a
nebuliser or an inhaler. Consequently, the invention may provide a nebuliser
of inhaler
comprising a composition of the invention.
A nebuliser is a device that converts liquid into aerosol droplets suitable
for inhalation.
Nebulisers use oxygen, compressed air or ultrasonic power to break up
medication
solutions and deliver a therapeutic dose of aerosol particles directly to the
lungs. A wide
variety of nebulisers is available. Nebulisers can be driven by compressed gas
(jet
nebuliser) or by an ultrasonically vibrating crystal (ultrasonic nebuliser).
In order to produce small enough particles from solution in 5-10 minutes, gas
flow rates of
at least 6 Liminute are usually necessary. Ultrasonic nebulisers use a rapidly
vibrating
piezoelectric crystal to produce aerosol particles. Ultrasonic nebuliser
machines are often
smaller and quieter.
Many nebulisers deliver only 10% of the prescribed drug dose to the lungs.
Much of the
drug is caught on the internal apparatus or wasted during exhalation. The
efficiency of drug
delivery depends on the type and volume of nebuliser chamber and the flow rate
at which it
is driven. Some chambers have reservoir and valve systems to increase
efficiency of
particle delivery during inspiration and reduce environmental losses during
expiration.
Breath-assisted open vent systems improve drug delivery but are dependent on
the patient
having an adequate expiratory flow. Face masks or mouthpieces may be used for
administration of aerosol particles.
Nebulisers are used for the treatment of many respiratory diseases.
Indications for
nebuliser use include the management of exacerbations and long-term treatment
of chronic
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obstructive pulmonary disease (COPD), management of cystic fibrosis,
bronchiectasis,
asthma, HIV/AIDS and symptomatic relief in palliative care.
Nebulised compositions of the invention may be used to prevent or treat a
microbial
infection, for example a microbial infection that comprises a biofilm, or a
microbe that is
5 capable of forming a biofilm, in a subject suffering from respiratory
disease, such as COPD,
cystic fibrosis, bronchiectasis, or asthma, or an HIV/AIDS-associated
respiratory infection,
or respiratory infection associated with terminal disease.
A composition of the invention may be used to prevent or treat a microbial
infection that
comprises a biofilm, or a microbe that is capable of forming a biofilm. The
biofilm may
10 comprise a biofilm-forming bacterium, fungus, or virus. The microbe that
is capable of
forming a biofilm may be a bacterium, fungus, or virus.
In some embodiments, a composition used to prevent or treat a microbial
infection may
exclude a nebulised composition of the invention.
The enzyme of a composition of the invention may be additional (i.e. added as
a result of
human intervention) to any enzyme activity able to convert the substrate to
release
hydrogen peroxide (referred to herein as "substrate conversion activity") that
may be
present in the substance, i.e. the composition may comprise the substance and
added
enzyme. In some embodiments there may be no substrate conversion activity in
the
substance.
A composition of the invention may be a storage-stable composition which does
not include
sufficient free water to allow the enzyme to convert the substrate.
For example, in some embodiments, the enzyme and the substance comprising a
substrate
for the enzyme may be encapsulated or contained within micelles (such as
reverse
micelles), and within the micelles there may not be sufficient free water to
allow the enzyme
to convert the substrate. A non-aqueous solvent, may be present in the
micelles.
Alternatively, compositions of the invention may be storage stable by virtue
of the enzyme
that is able to convert a substrate to release hydrogen peroxide and the
substance that
includes a substrate for the enzyme, being separate (or compartmentalised)
from water in
the composition. For example, the composition may be a double emulsion.
Droplets
containing the enzyme and the substance (but without sufficient free water to
allow the
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enzyme to convert the substrate) may be dispersed within globules of oil, and
the globules
of oil may be dispersed within an aqueous phase (e.g. water).
In the presence of sufficient water, the enzyme of the storage-stable
composition is able to
convert the substrate and release hydrogen peroxide. Hydrogen peroxide is
known to be
effective against a wide variety of different microbes. Thus, antimicrobial
activity is
generated following dilution of a storage-stable composition of the invention.
If a storage-stable composition is used, this may be diluted by liquid present
at the site of
administration leading to release of hydrogen peroxide at the administration
site.
Compositions of the invention that do not include water, or any free water,
may provide
particularly stable compositions of the invention, since the enzyme will not
be able to
convert the substrate to release hydrogen peroxide until the composition is
contacted with
sufficient amount of water.
Catalase is an enzyme that catalyses the decomposition of hydrogen peroxide to
water and
oxygen. The use of a substance that lacks catalase activity means that there
is no
variability in the amount of this activity between similar substances from
different sources,
or from different harvests from the same source. This reduces the variability
in antimicrobial
activity that can be generated from such substances. Alternatively, if the
substance does
include catalase activity, and it is not possible or desirable to inactivate
the catalase activity
in the substance prior to contacting the substance with the enzyme, then
sufficient enzyme
may be used such that the effect of catalase activity on the hydrogen peroxide
that can be
generated from the substance is reduced. This also reduces the variability in
antimicrobial
activity that can be generated from the substance. In some embodiments, the
substance
may lack catalase activity.
Catalase is present in many plants and animals. Catalase activity may be
removed during
processing or extraction of the substance, or inactivated before use of the
substance in the
composition. Catalase activity may be heat inactivated, for example by
pasteurisation. A
suitable temperature for heat inactivation of catalase activity is at least 60
C, 70 C, or 80 C,
preferably for at least 2 minutes.
The term "storage-stable" is used herein to mean that the composition can be
stored at
ambient temperature for at least several days, suitably at least a week or at
least one or
two months, whilst retaining the ability to generate antimicrobial activity
following dilution of
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the composition. The storage temperature may be below 37 C, preferably 20-25
C.
Preferably compositions are stored away from exposure to light.
Hydrogen peroxide is generally unstable at ambient temperature. The lack of
sufficient free
water in a storage-stable composition of the invention prevents the enzyme
converting the
substrate to release hydrogen peroxide, and thus helps to maintain the
stability of the
composition for extended periods at ambient temperature. A storage-stable
composition of
the invention may include some water provided that there is not sufficient
free water to
allow the enzyme to convert the substrate. Suitable amounts of water will vary
depending
on the precise components of the composition. However, typically, a storage-
stable
composition of the invention comprises less than 20% total water content, for
example,
10%-19%, water.
Hydrogen peroxide may be released for a sustained period following dilution of
the
composition, depending on the amount of substrate present in the composition,
and the
activity of the enzyme. It will be appreciated that the amount of substrate
and/or the activity
of enzyme in the composition may be selected to provide for release of a
relatively high
level of hydrogen peroxide for a short period, or for release of a lower level
of hydrogen
peroxide for a longer period, following dilution of the composition. Suitably
the composition
provides for sustained release of hydrogen peroxide for a period of at least
twenty four
hours, more preferably at least forty eight hours, following dilution of the
composition.
Suitably the composition provides for sustained release of hydrogen peroxide
at a level of
less than 2 mmol/litre for a period of at least twenty four hours, following
dilution of the
composition.
A composition of the invention may comprise sufficient enzyme and substrate to
provide for
sustained release of at least 0.1, 0.5, 1 or 1.5 mmol/litre hydrogen peroxide
for a period of
at least 24 hours, more preferably 48 hours.
It will be appreciated that there should be sufficient enzyme present in a
storage-stable
composition of the invention to convert the substrate and form hydrogen
peroxide as
needed following dilution of the composition.
In view of the importance of generation of hydrogen peroxide by storage-stable
compositions of the invention in the presence of sufficient water, it will be
appreciated that
the compositions should not contain any added peroxidase.
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In some embodiments the enzyme is a purified enzyme. The term "purified
enzyme" is
used herein to include an enzyme preparation in which the enzyme has been
separated
from at least some of the impurities originally present when the enzyme was
produced.
Preferably impurities that have been removed or reduced include those that
would
otherwise interfere with the ability of the enzyme to convert the substrate to
release
hydrogen peroxide.
It may not always be necessary or desirable that the purified enzyme is at a
high level of
purity provided that the enzyme is able to convert the substrate to release
hydrogen
peroxide. In some circumstances, it may be desirable to use a relatively crude
enzyme
preparation. Examples of suitable purity levels include at least 10%, 20%,
30%, 40%, 50%,
60%, 70%, 80%, or 90% pure.
It is preferred, however, that the amount of any catalase that may originally
have been
present when the enzyme was produced has been reduced. The enzyme may have
been
produced by recombinant or non-recombinant means, and may be a recombinant or
non-
recombinant enzyme. The enzyme may be purified from a microbial source,
preferably from
a non-genetically modified microbe.
The level of purity of the enzyme may be selected as appropriate depending on
the
intended use of the composition. For example, if the composition is intended
for medical
use, a medical grade or medical device grade of purity should be used.
Thus, there is provided according to the invention a storage-stable
composition for
generating antimicrobial activity, which comprises: a lipophilic phase; an
aqueous phase; a
purified enzyme that is able to convert a substrate to release hydrogen
peroxide; and a
substance that includes a substrate for the enzyme; wherein the composition
does not
include sufficient free water to allow the enzyme to convert the substrate.
According to the invention, there is provided a storage-stable composition for
generating
antimicrobial activity, comprising: a first phase; a second phase; an enzyme
that is able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a
substrate for the enzyme, wherein the first phase and the second phase are
immiscible,
and wherein the composition does not comprise sufficient free water to allow
the enzyme to
convert the substrate.
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According to the invention, there is provided a storage-stable composition for
generating
antimicrobial activity, which comprises: an oil; an emulsifier; an enzyme that
is able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a
substrate for the enzyme, wherein the composition does not include sufficient
free water to
allow the enzyme to convert the substrate.
In some embodiments the enzyme is an oxidoreductase enzyme. Examples of
oxidoreductase enzymes that can convert a substrate to release hydrogen
peroxide include
glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase,
pyranose
oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase, and amioacid
oxidase. The
corresponding substrates for these oxidoreductase enzymes are D-glucose,
hexose,
cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate and
aminoacid,
respectively.
A mixture of one or more oxidoreductase enzymes and one or more substrates for
the
oxidoreductase enzymes may be present in a composition of the invention.
The oxidoreductase enzyme may be glucose oxidase, and the substrate may be D-
glucose.
The substance may be any substance that includes a substrate for the enzyme.
In some
embodiments the substance lacks catalase activity.
Thus, there is also provided according to the invention a storage-stable
composition for
generating antimicrobial activity, which comprises: a lipophilic phase; an
aqueous phase;
an enzyme that is able to convert a substrate to release hydrogen peroxide;
and a
substance that lacks catalase activity and that includes a substrate for the
enzyme; wherein
the composition does not include sufficient free water to allow the enzyme to
convert the
substrate.
There is also provided, according to the invention, a storage-stable
composition for
generating antimicrobial activity, comprising: a first phase; a second phase;
an enzyme that
is able to convert a substrate to release hydrogen peroxide; and a substance
that lacks
catalase activity and that includes a substrate for the enzyme, wherein the
first phase and
the second phase are immiscible, and wherein the composition does not comprise
sufficient free water to allow the enzyme to convert the substrate.
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There is also provided according to the invention a storage-stable composition
for
generating antimicrobial activity, which comprises: an oil; an enzyme that is
able to convert
a substrate to release hydrogen peroxide; and a substance that lacks catalase
activity and
that includes a substrate for the enzyme, and wherein the composition does not
include
5 sufficient free water to allow the enzyme to convert the substrate.
The substance may be an unrefined substance. The term "unrefined" is used
herein to refer
to substances that have not been processed into a pure form. Unrefined
substances
include substances that may have been concentrated, for example by drying or
boiling.
The substance may include one or more substrates from a natural source (termed
herein a
10 "natural substance"). Examples of natural substances include substances
from a plant
source, including from sap, roots, nectar, flowers, seeds, fruit, leaves, or
shoots. The
substance may be an unrefined natural substance.
Suitably the substance comprises one or more of the following substrates: D-
glucose,
hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate or
amino acid.
15 The substance may be a sugar substance. The term "sugar substance" is
used herein to
mean any substance that includes one or more sugars. The term "sugar is used
herein to
refer to a carbohydrate with the general formula Cm(H2O)n. Preferred sugars
include
monosaccharides, such as D-glucose, hexose, or D-galactose. The sugar
substance may
include one or more sugars from a natural source (termed herein a "natural
sugar
substance"). The natural sugar substance may be an unrefined natural sugar
substance.
The unrefined natural sugar substance may be (or be derived from) a natural
sugar
product. In some embodiments, the unrefined natural sugar product is a honey.
In some
embodiments, the honey is a honey that has been treated to remove or
inactivate catalase
activity.
As discussed above, the substance itself may preferably lack an enzyme
activity that is
able to convert the substrate to release hydrogen peroxide (referred to as
"substrate
conversion activity"). Absence of substrate conversion activity from the
substance has the
advantage that there is then no variability in the amount of this activity
between similar
substances from different sources, or from different harvests from the same
source. This
further reduces the variability in antimicrobial activity that can be
generated from such
substances. Substrate conversion activity is then provided only by the enzyme
that is
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16
contacted with the substance, and so the amount of substrate conversion
activity present in
the composition can be controlled.
Substrate conversion activity may be removed during processing or extraction
of the
substance, or inactivated before use of the substance in a composition of the
invention.
Substrate conversion activity may be inactivated by heat inactivation, for
example by
pasteurisation. A suitable temperature for heat inactivation of substrate
conversion activity
is at least 80 C, preferably for at least two minutes. An advantage of heat
inactivation is
that both catalase activity and substrate conversion activity can be
inactivated in a single
heat inactivation step.
In some embodiments of the invention, the substance is a processed, extracted,
or refined
substance (i.e. a substance in which impurities or unwanted elements have been
removed
by processing). Preferably impurities that have been removed or reduced
include those that
would otherwise interfere with the ability of the enzyme to convert the
substrate to release
hydrogen peroxide.
In some embodiments of the invention, the substance comprises a purified
substrate for the
enzyme. The term "purified substrate" is used herein to include a substrate
preparation in
which the substrate has been separated from at least some of the impurities
originally
present when the substrate was obtained or produced. The purified substrate
may be
obtained from a natural source or may be synthetically produced. The purified
substrate
may be a processed, extracted, or refined substrate (i.e. a substrate in which
impurities or
unwanted elements have been removed by processing).
It may not always be necessary or desirable that the purified substrate is at
a high level of
purity provided that the enzyme is able to convert the substrate to release
hydrogen
peroxide. In some circumstances, it may be desirable to used a relatively
crude substrate
preparation. Examples of suitable purity levels include at least 10%, 20%,
30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or 99% pure. However, in some embodiments, it may be
desirable that the purified substrate is a medical grade, medical device
grade, or
pharmaceutical grade substrate.
In particular embodiments, the purified substrate is or comprises a purified
sugar
substance. The purified sugar substance may be obtained from a natural source
(for
example a processed, extracted, or refined natural sugar substance), or be
synthetically
produced. The purified sugar substance may be at least 10%, 20%, 30%, 40%,
50%, 60%,
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70%, 80%, 90%, 95%, or 99% pure. The purified sugar substance may be a medical
grade,
medical device grade, or pharmaceutical grade sugar substance,. The purified
sugar
substance may include one or more purified sugar substances, for example
purified D-
glucose, hexose, or D-galactose. For example the purified sugar substance may
be
medical grade, medical device grade, or pharmaceutical grade D-glucose,
hexose, or D-
galactose.
There is also provided according to the invention a composition for generating
anti-
microbial activity, wherein the composition comprises: a lipophilic phase; an
aqueous
phase; an enzyme that is able to convert a substrate to release hydrogen
peroxide; and a
substance that includes a purified substrate for the enzyme.
There is also provided, according to the invention, a composition for
generating
antimicrobial activity, comprising: a first phase; a second phase; an enzyme
that is able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a purified
substrate for the enzyme, wherein the first phase and the second phase are
immiscible.
There is also provided according to the invention a composition for generating
anti-
microbial activity, wherein the composition comprises: an oil; an enzyme that
is able to
convert a substrate to release hydrogen peroxide; and a substance that
includes a purified
substrate for the enzyme.
The composition may be a storage-stable composition for generating
antimicrobial activity,
which comprises: a lipophilic phase; an aqueous phase; an enzyme that is able
to convert
a substrate to release hydrogen peroxide; and a substance that includes a
purified
substrate for the enzyme; wherein the composition does not include sufficient
free water to
allow the enzyme to convert the substrate.
The composition may be a storage-stable composition for generating
antimicrobial activity,
comprising: a first phase; a second phase; an enzyme that is able to convert a
substrate to
release hydrogen peroxide; and a substance that includes a purified substrate
for the
enzyme, wherein the first phase and the second phase are immiscible, and
wherein the
composition does not comprise sufficient free water to allow the enzyme to
convert the
substrate.
The composition may be a storage-stable composition for generating
antimicrobial activity,
which comprises: an oil; an enzyme that is able to convert a substrate to
release hydrogen
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peroxide; and a substance that includes a purified substrate for the enzyme,
and wherein
the composition does not include sufficient free water to allow the enzyme to
convert the
substrate.
The composition may be a storage-stable composition for generating
antimicrobial activity,
which comprises: a lipophilic phase; an aqueous phase; an enzyme that is able
to convert
a substrate to release hydrogen peroxide; and a substance that lacks catalase
activity and
that includes a purified substrate for the enzyme; wherein the composition
does not include
sufficient free water to allow the enzyme to convert the substrate.
The composition may be a storage-stable composition for generating
antimicrobial activity,
comprising: a first phase; a second phase; an enzyme that is able to convert a
substrate to
release hydrogen peroxide; and a substance that lacks catalase activity and
that includes a
substrate for the enzyme, wherein the first phase and the second phase are
immiscible,
and wherein the composition does not include sufficient free water to allow
the enzyme to
convert the substrate.
The composition may be a storage-stable composition for generating
antimicrobial activity,
which comprises: an oil; an enzyme that is able to convert a substrate to
release hydrogen
peroxide; and a substance that lacks catalase activity and that includes a
purified substrate
for the enzyme; wherein the composition does not include sufficient free water
to allow the
enzyme to convert the substrate.
In particular embodiments, the enzyme and the substrate are purified, for
example purified
glucose oxidase and purified D-glucose, suitably medical grade, medical device
grade, or
pharmaceutical grade glucose oxidase and D-glucose.
The ratio of the lipophilic phase to the aqueous phase, or the ratio of the
first phase to the
second phase, in a composition of the invention may be from 9:1 to 1:9, 8:1 to
1:8, 7:1 to
1:7, 6:1 to 1:6, 5:1 to 1:5, 4:1 to 1:4, 3:1 to 1:3, or 2:1 to 1:2 (v/v), for
example from 4:1 to
1:4.
A composition of the invention may comprise 5-95%, 10-95%, 15-95%, 20-95%, 25-
95%,
30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-
95%,
80-95%, 85-95%, or 90-95% (v/v) lipophilic phase, or first phase (including
any emulsifying
agent present).
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Alternatively, a composition of the invention may comprise 5-95%, 5-90%, 5-
85%, 5-80%,
5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-
20%, 5-15%, or 5-10% (v/v) lipophilic phase, or first phase (including any
emulsifying agent
present).
A composition of the invention may comprise 5-95%, 10-95%, 15-95%, 20-95%, 25-
95%,
30-95%, 35-95%, 40-95%, 45-95%, 50-95%, 55-95%, 60-95%, 65-95%, 70-95%, 75-
95%,
80-95%, 85-95%, or 90-95% (v/v) aqueous phase, or second phase.
Alternatively, a composition of the invention may comprise 5-95%, 5-90%, 5-
85%, 5-80%,
5-75%, 5-70%, 5-65%, 5-60%, 5-55%, 5-50%, 5-45%, 5-40%, 5-35%, 5-30%, 5-25%, 5-
20%, 5-15%, or 5-10% (v/v) aqueous phase, or second phase.
A composition of the invention may comprise 1-60%, 1-50%, 1-40%, 1-30%, 1-20%,
or 1-
10% (w/v) of the substance, for example a honey.
A composition of the invention may comprise 1-60%, 5-60%, 10-60%, 15-60%, 20-
60%,
25-60%, 30-60%, 35-60%, 40-60%, 45-60%, or 50-60% (w/v) of the substance, for
example a honey.
A composition of the invention may comprise 1-1500 units, 15-1500 units, 30-
1500 units,
50-1500 units, 100-1500 units, 1-<685 units, 15-<685 units, 30-<685 units, 50-
<685 units,
100-<685 units, 500-1000 units, 685-1000 units, or 100-500 units, of the
enzyme,
preferably glucose oxidase, per gram of the composition.
A composition of the invention may comprise no more than 85% water, for
example no
more than 80%, 70%, 60%, 50%, 40%, 30%, or 20% water, or less than 20% water,
for
example 10-19% water. A composition of the invention may comprise less than
20% (w/w).
A composition of the invention may comprise less than 15% (w/w) water. A
composition of
the invention may comprise less than 12% (w/w) water.
A composition of the invention may comprise 10-60% (w/w) of non-aqueous
solvent. In
some embodiments, a composition of the invention may comprise 20-50% (w/w) of
a non-
aqueous solvent. In some embodiments, a composition of the invention may
comprise 35-
40% (vv/w) of a non-aqueous solvent.
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A composition of the invention may comprise 10-40 % (w/w) of the first phase,
e.g.
lipophilic phase (such as oil). The composition may comprise 20-30% (w/w) of
the first
phase, e.g. lipophilic phase (such as oil).
A composition of the invention may comprise 1-10% (w/w) emulsifier. The
composition may
5 comprise 1-5% (w/w) emulsifier. The emulsifier is preferably a
surfactant.
A composition of the invention may comprise 10-50% (w/w) of the substance
which
comprises a substrate for the enzyme. The composition may comprise 20-40%
(w/w) of the
substance. In some embodiments, the composition may comprise 25 to 35% (w/w)
of the
substance.
10 A composition of the invention may comprise 20-50% (w/w) of non-aqueous
solvent, 20-
30% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1-5% (w/w)
emulsifier and
20-40% (w/w) of the substance which comprises a substrate for the enzyme.
A composition of the invention may comprise 10-60% (w/w) of non-aqueous
solvent, 10-
40% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1-10% (w/w)
emulsifier and
15 10-50% (w/w) of the substance which comprises a substrate for the
enzyme.
A composition of the invention may comprise 35-45% (w/w) of non-aqueous
solvent, 20-
30% (w/w) of the first phase e.g. lipophilic phase (such as oil), 1-5% (w/w)
emulsifier and
25-35% (w/w) of the substance which comprises a substrate for the enzyme.
A composition of the invention may comprise 30-60% (v/v) solvent, such as a
non-
20 aqueous, polar solvent.
A composition of the invention may comprise 30-60% (v/v) first phase, such as
a lipophilic
phase (e.g. oil),
A composition of the invention may comprise 1-10% (v/v) emulsifier such e.g.
surfactant,
A composition of the invention may comprise 30-70% or 40-60% (w/w) of the
substance
that comprises a substrate for the enzyme, for example honey.
The ratio of the first phase to the second phase in a composition of the
invention may be
51:1 (v/v), for example 0,1-1:1 (v/v). In some embodiments, the ratio of the
first phase to
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the second phase is <0.6:1 (v/v), for example 0.1-<0.6:1 (v/v). In some
embodiments, the
ratio of the first phase to the second phase is Q.4:1 (v/v), for example 0.1-
0.4:1 (v/v).
The first phase in a composition of the invention may be present at less than
60% (v/v) of
the composition. In some embodiments, the first phase is present at 10% to
less than 60%
(v/v) of the composition. In some embodiments, the first phase is present at
10% to less
than 50% (v/v) of the composition. In some embodiments, the first phase is
present at 10%
to less than 40% (v/v) of the composition. In some embodiments, the first
phase is present
at 10% to less than 30% (v/v) of the composition. In some embodiments, the
first phase is
present at 10% to less than 25% (v/v) of the composition.
A composition of the invention may comprise an emulsifier. In some
embodiments, the
emulsifier is present at up to 25% (v/v) of the composition, for example 1-25%
(v/v) of the
composition, 5-25% (v/v) of the composition, or 10-25% (v/v) of the
composition.
The ratio of the amount of the substance that includes a substrate for the
enzyme to the
volume of the second phase in a composition of the invention may be from 0.5:1
to 2:1, for
example 1:1.
The amount of the substance that includes a substrate for the enzyme in a
composition of
the invention may be up to 70% (w/v) of the composition, for example 5-70%
(w/v), 10-70%
(w/v), 20-70% (w/v), or 30-70% (w/v), or up to 60% (w/v) of the composition,
for example 5-
60% (w/v), 10-60% (w/v), 20-60% (w/v), or 30-60% (w/v), of the composition.
A composition of the invention may be an emulsion. In particular embodiments,
a
composition of the invention is an emulsion that comprises reverse micelles.
The reverse
micelles may be formed by the second phase.
In some embodiments of a composition of the invention, the enzyme and the
substance
that includes a substrate for the enzyme is dissolved in the second phase.
In particular embodiments of the invention, the first phase is, or comprises
paraffin oil.
In particular embodiments of the invention, the second phase is, or comprises
glycerol.
In particular embodiments of the invention, the emulsifier is, or comprises
Polyglycerol
polyricinoleate (PGPR).
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In particular embodiments of the invention, the enzyme that is able to convert
a substrate to
release hydrogen peroxide is, or comprises purified glucose oxidase, and the
substance
that includes a substrate for the enzyme is, or comprises honey.
In other particular embodiments of the invention, the enzyme that is able to
convert a
substrate to release hydrogen peroxide is, or comprises purified glucose
oxidase, and the
substance that includes a substrate for the enzyme is, or comprises purified
glucose.
In some embodiments, a composition of the invention is a cream. Typically, the
viscosity of
an emulsion used as a cream will be higher than that of an emulsion used as a
spray. A
cream may be formed by including a viscosity-increasing agent, such as a
thickener or
gelling agent (for example a hydrocolloid) in the composition.
Hydrocolloids are a heterogeneous group of hydrophilic, long-chain polymers
(polysaccharides or proteins) characterised by their ability to form viscous
dispersions
and/or gels when dispersed in water (Saha and Bhattacharya, J Food Sci
Technol, 2010,
47(6):587-597). The extent of thickening varies with the type and nature of
the hydrocolloid.
Some provide low viscosities at a fairly high concentration, but most provide
a high
viscosity at a concentration below 1%. The viscosity of hydrocolloid
dispersions arises
predominantly from non-specific entanglement of conformationally disordered
polymer
chains. Hydrocolloids that can be used as thickening agents (referred to
herein as
hydrocolloid thickeners) include starch, modified starch, xanthan,
galactomannans (such as
guar gum, locust bean gum, and tara gum), gum Arabic or acacia gum, gum
karaya, gum
tragacanth, konjac maanan, and cellulose derivatives such as carboxymethyl
cellulose,
methyl cellulose, and hydroxypropylmethyl cellulose.
Some hydrocolloids are able to form gels, consisting of polymer molecules
cross-linked to
form an interconnected molecular network immersed in a liquid medium. A
rheological
definition of a gel is a viscoelastic system with a 'storage modulus' (G")
larger than the 'loss
modulus' (G") (de Vries 2004, Gums and stabilizers for the food industry, vol
12. RSC Publ,
Oxford, pp 22-30). Hydrocolloids form gels by physical association of their
polymer chains
through hydrogen bonding, hydrophobic association, and cation-mediated cross-
linking.
Gelling-type hydrocolloids (or hydrocolloid gelling agents) include alginate,
pectin,
carrageenan, gelatin, gellan, agar, modified starch, methyl cellulose and
hydroxypropylmethyl cellulose.
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Gelation of hydrocolloids can occur by different mechanisms: ionotropic
gelation, cold-set
gelation and heat-set gelation (Burey et al. 2008, Crit Rev Food Sci Nutr
48:361-377).
lonotropic gelation occurs via cross-linking of hydrocolloid chains with ions,
typically a
cation-mediated gelation process of negatively-charged polysaccharides.
Examples of
hydrocolloids that can form gels by ionotropic gelation include alginate,
carrageenan and
pectin. lonotropic gelation can be carried out by either diffusion setting or
internal gelation.
In cold-set gelation, hydrocolloid powders are dissolved in warm/boiling water
to form a
dispersion which forms a gel on cooling. Agar and gelatin form gels by this
mechanism.
Heat-set gels require the application of heat to gel (for example, curdlan,
konjac
glucomannan, methyl cellulose, starch and globular proteins).
Thus, in some embodiments, a composition of the invention comprises a
viscosity-
increasing agent, such as a thickener or gelling agent, for example a
hydrocolloid. In
particular embodiments, the hydrocolloid is, or comprises, a polysaccharide or
a protein.
The hydrocolloid may be a hydrocolloid thickener, such as starch, modified
starch, xanthan,
a galactomannan (such as guar gum, locust bean gum, and tara gum), gum Arabic
or
acacia gum, gum karaya, gum tragacanth, konjac maanan, or a cellulose
derivative, such
as carboxymethyl cellulose, methyl cellulose, or hydroxypropylmethyl
cellulose.
In other embodiments, the hydrocolloid is, or comprises a cross-linked
hydrocolloid, for
example a cross-linked polysaccharide, such as cross-linked alginate, pectin,
carrageenan,
gelatin, gellan, agar, agarose, modified starch, or a cellulose derivative,
such as methyl
cellulose or hydroxypropylmethyl cellulose.
The hydrocolloid may be cross-linked by any suitable method, for example
including the
methods for gelation of hydrocolloids described above: ionotropic gelation,
cold-set gelation
and heat-set gelation. In particular embodiments, molecules of the
hydrocolloid are cross-
linked by cations (for example calcium ions) as a result of ionotropic
gelation of a
hydrocolloid gelling agent. Examples of hydrocolloid cross-linked by cations
that may be
present in a composition of the invention include alginate, carrageenan or
pectin.
In particular embodiments, a composition of the invention includes cross-
linked alginate, for
example alginate cross-linked by calcium ions. Alginate can form gels without
prior heating
because sodium alginate is soluble in cold water.
Cross-linked alginate may be formed from sodium alginate and calcium ions (for
example,
provided by calcium chloride). In some embodiments, water may be used as
solvent to
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dissociate the calcium ions. However, since this could potentially activate
production of
hydrogen peroxide by the enzyme and the substance that includes a substrate
for the
enzyme, and limit the stability of the composition, it may be preferred to use
a non-aqueous
solvent to dissociate the calcium ions, such as ethanol or acetic acid.
We have appreciated that glycerol may be used to bind free water. This
property allows water
to be used to dissolve the alginate, provided sufficient glycerol is present
to prevent premature
release of hydrogen peroxide from the enzyme and the substance that includes a
substrate for
the enzyme.
There is also provided according to the invention a method of making a
composition of the
invention, which comprises mixing a lipophilic component, an aqueous
component, an
enzyme that is able to convert a substrate to release hydrogen peroxide, and a
substance
that includes a substrate for the enzyme to form the composition.
There is also provided, according to the invention, a method of making a
composition of the
invention, comprising mixing a first component (or liquid of a first phase), a
second
component (or liquid of a second phase), an enzyme that is able to convert a
substrate to
release hydrogen peroxide, and a substance that includes a substrate for the
enzyme to
form the composition, wherein the first component (or liquid of the first
phase) and second
component (or liquid of the second phase) are immiscible.
There is also provided according to the invention a method of making a
composition of the
invention, which comprises mixing an oil, an enzyme that is able to convert a
substrate to
release hydrogen peroxide, and a substance that includes a substrate for the
enzyme to
form the composition.
Methods of the invention may also comprise mixing a non-aqueous solvent, such
as a
polar, organic solvent.
A method of the invention may employ a rheometer to form compositions of the
invention.
A rheometer may allow for control of shear rate and temperature.
The enzyme, and the substance comprising a substrate for the enzyme may be
dissolved
in a non-aqueous solvent to form a first mixture. The emulsifier may be added
to the first
phase, lipophilic phase or oil to form a second mixture. The first mixture may
then be added
dropwise to the second mixture to form an emulsion, whilst being mixed using
e.g. a
rheometer or mixer.
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To form an emulsion, mixing may occur at a shear rate of between 1500 1/s to
2500 1/s,
such as 2000 1/s. Mixing may occur at 30 and 50 C, e.g. about 37 C.
There is further provided according to the invention a method of making a
composition of
the invention, which comprises mixing the enzyme, the substance that includes
a substrate
5 for the enzyme, liquid of the second phase, liquid of the first phase,
and optionally an
emulsifier, under a high rate of shear for sufficient time to form an
emulsion.
More stable emulsions may be formed if the ingredients of the emulsion are pre-
mixed
before contact with the emulsifier. Thus, in some embodiments, the enzyme, the
substance
that includes a substrate for the enzyme, liquid of the second phase, and
liquid of the first
10 phase are pre-mixed under a high rate of shear before contacting the pre-
mixed ingredients
with the emulsifier and mixing of the mixture comprising the pre-mixed
ingredients and the
emulsifier under the high rate of shear.
In some embodiments, the enzyme and the substance that includes a substrate
for the
enzyme are dissolved in the liquid of the second phase to form a solution
before contacting
15 the solution with the liquid of the first phase.
The high rate of shear may be from 1000 1/s to 4000 1/s. We have found that
emulsions
made using methods of the invention are more stable when formed using a higher
rate of
shear, for example from >2000 1/s to 4000 its, >2000 1/s to 3500 1/s, >2500
Its to 4000
1/s, or >2500 1/s to 3500 1/s.
20 Mixing of the enzyme, the substance that includes a substrate for the
enzyme, liquid of the
second phase, liquid of the first phase, and the emulsifier (if present) may
be carried out at
a temperature of 20 C to 40 C, for example from 35 C to 40 C. More stable
emulsions may
be formed when mixing of the enzyme, the substance that includes a substrate
for the
enzyme, liquid of the second phase, liquid of the first phase, and the
emulsifier (if present)
25 is carried out at higher temperatures, for example from >37.5 C to 40 C,
or 38 C to 40 C.
In some embodiments of methods of the invention, the enzyme, the substance
that
includes a substrate for the enzyme, liquid of the second phase, liquid of the
first phase,
and the emulsifier (if present), are mixed under a high rate of shear for at
least 5 minutes,
for example for 5 to 30 minutes.
Methods of the invention may be used to form creams, for example by inclusion
of a
viscosity-increasing agent, such as a thickener or gelling agent, for example
a hydrocolloid.
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In some embodiments, a method of the invention further comprises mixing a
viscosity
increasing agent with the enzyme, the substance that includes a substrate for
the enzyme,
liquid of the second phase, liquid of the first phase, and the emulsifier (if
present), under
the high shear rate to form a cream.
In some embodiments, the viscosity-increasing agent is, or comprises a
hydrocolloid, for
example a polysaccharide.
In some embodiments, the hydrocolloid is, or comprises a hydrocolloid
thickener, such as
starch, modified starch, xanthan, a galactomannan (such as guar gum, locust
bean gum,
and tam gum), gum Arabic or acacia gum, gum karaya, gum tragacanth, konjac
maanan,
or a cellulose derivative, such as carboxymethyl cellulose, methyl cellulose,
or
hydroxypropylmethyl cellulose.
In some embodiments, the hydrocolloid is, or comprises a hydrocolloid gelling
agent, such
as alginate, pectin, carrageenan, gelatin, gellan, agar, agarose, modified
starch, or a
cellulose derivative, such as methyl cellulose or hydroxypropylmethyl
cellulose.
The hydrocolloid gelling agent may capable of forming a gel by ionotropic
gelation in the
presence of cations. In such embodiments, a method of the invention further
comprises
mixing the cations with the hydrocolloid gelling agent, the enzyme, the
substance that
includes a substrate for the enzyme, liquid of the second phase, liquid of the
first phase,
and the emulsifier (if present), under the high shear rate to form the cream.
In some embodiments, the hydrocolloid gelling agent capable of forming a gel
by ionotropic
gelation, the enzyme, the substance that includes a substrate for the enzyme,
liquid of the
second phase, liquid of the first phase, and the emulsifier (if present), are
mixed to form a
mixture prior to contacting the cations with the mixture.
In some embodiments the hydrocolloid gelling agent capable of forming a gel by
ionotropic
gelation is contacted with the liquid of the second phase, the enzyme, and the
substance
that includes a substrate for the enzyme, prior to contact with the liquid of
the first phase.
In some embodiments the cations are, or comprise calcium ions.
In particular embodiments the hydrocolloid gelling agent capable of forming a
gel by
ionotropic gelation in the presence of cations is, or comprises alginate,
carrageenan or
pectin, for example alginate.
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In a particular embodiment, the hydrocolloid gelling agent is provided in
aqueous solution,
and the second phase is glycerol, wherein the glycerol is present in
sufficient amount to
bind free water in the composition and thereby prevent the enzyme catalysing
release of
hydrogen peroxide from the substance that includes a substrate for the enzyme.
In some embodiments the hydrocolloid gelling agent that is able to form a gel
by ionotropic
gelation (for example, alginate) is pre-mixed with the substance that includes
a substrate
for the enzyme, liquid of the second phase, and liquid of the first phase
under a high rate of
shear before the pre-mixed ingredients are contacted with the emulsifier (if
present) and
the cations (for example, calcium ions), and the mixture comprising the pre-
mixed
ingredients, the emulsifier (if present), and the cations is mixed under the
high rate of
shear.
The emulsifier (if present) may be contacted with the pre-mixed ingredients
before the
cations (for example, calcium ions). The cations (for example, calcium ions)
may be added
dropwise.
The cations (for example, calcium ions) may be provided in aqueous solution.
Alternatively,
the cations may be provided in non-aqueous solution, using a non-aqueous
solvent, such
as ethanol or acetic acid.
In particular embodiments, calcium chloride is provided in ethanol, and sodium
alginate is
provided in aqueous solution, and the second phase is glycerol, and the
glycerol is present
in sufficient amount to bind the free water in the alginate solution and
thereby prevent the
enzyme catalysing release of hydrogen peroxide from the substance that
includes a
substrate for the enzyme. This prevents premature release of hydrogen peroxide
until the
composition is contacted with water, thereby providing a stable composition.
As above, a method of the invention may employ a rheometer to form a
composition of the
invention. A rheometer may allow for control of shear rate and temperature.
Alternatively, a
high rate of shear may be provided by use of an ultrasonic probe, or a
homogeniser.
There is also provided according to the invention, a pharmaceutical
composition comprising
a composition of the invention and a pharmaceutically acceptable carrier,
excipient or
diluent.
There is also provided according to the invention a composition of the
invention for use as
a medicament.
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There is further provided according to the invention a composition of the
invention for use
in the prevention or treatment of a microbial infection.
The prevention or treatment is preferably by topical administration of the
composition. The
prevention or treatment may be by administration to a subject's respiratory
tract. The
prevention or treatment may be by administration to the body cavity. The
prevention or
treatment may be by internal administration to a subject.
There is also provided according to the invention a method of prevention or
treatment of a
microbial infection, which comprises administering an effective amount of a
composition of
the invention to a subject in need of such treatment.
The invention also provides use of a composition of the invention in the
manufacture of a
medicament for the prevention or treatment of a microbial infection.
The microbial infection may be a viral infection, for example, a herpes
simplex virus (HSV)
infection. In other embodiments, the microbial infection may be a fungal
infection, or a
bacterial infection.
The microbial infection may be: a nasal infection, such as sinusitis or
rhinosinusitis; a
respiratory tract infection, such as an upper respiratory tract infection
(e.g. tonsillitis,
laryngitis or sinusitis) or a lower respiratory tract infection (e.g.
bronchitis, pneumonia,
bronchiolitis or tuberculosis); an infection associated with chronic
obstructive pulmonary
disease (COPD), cystic fibrosis, bronchiectasis, asthma, or an HIV/AIDS-
associated
respiratory infection, or a respiratory infection associated with terminal
disease.
A composition of the invention may be used to prevent or treat a microbial
infection that
comprises a biofilm, or a microbe that is capable of forming a biofilm. The
biofilm may
comprise a biofilm-forming bacterium, fungus, or virus. The microbe that is
capable of
forming a biofilm may be a bacterium, fungus, or virus.
A composition of the invention may be used in an antimicrobial wipe, in
disinfection, for
example in hospital disinfection, or as an antimicrobial spray, for example as
a topical
prophylactic antimicrobial spray to disinfect part of the body of a patient
prior to surgery.
A composition of the invention may include an antimicrobial agent. For
example, hydrogen
peroxide may be present if the composition is formed by contacting the enzyme
with the
substance in aqueous solution under conditions for conversion of the substrate
by the
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enzyme, and then drying the composition to reduce its water content to a level
where there
is insufficient free water to allow the enzyme to convert the substrate.
Preferably, however,
the composition does not include any detectable hydrogen peroxide. Such
composition
may be formed, for example, by contacting the enzyme with the substrate in the
absence of
sufficient free water to allow the enzyme to convert the substrate. Examples
of other
antimicrobial agents that may be present in a storage-stable composition of
the invention
include: an antibiotic, an antiviral agent, or an anti-fungal agent.
The composition may be a medical grade or medical device grade composition, or
a
pharmaceutical grade composition.
Each component of the composition may be a natural substance (i.e. each
component is
derived or purified from a natural source). Compositions for use according to
the invention
which contain only natural ingredients provide an attractive alternative to
drug-based
antimicrobial formulations.
Advantageously the substance is a honey. The honey may be a medical grade or
medical
device grade honey. In some embodiments, the honey is a honey that has been
treated to
remove or inactivate catalase activity originally present in the honey.
According to an
embodiment of the invention, the substance is a pasteurised honey, and the
enzyme is a
glucose oxidase. According to some embodiments, the substance is a medical
grade or
medical device grade honey, and the enzyme is a medical grade or medical
device grade
enzyme, suitably glucose oxidase.
Honey is a natural product made by honey bees using nectar from flowers. It is
a saturated
or super-saturated solution of sugars. Honey is defined in the Codex
Alimentarius
international food standard as "the natural sweet substance produced by honey
bees from
the nectar of plants or from secretions of living parts of plants or
excretions of plant sucking
insects on the living parts of plants, which the bees collect, transform by
combining with
specific substances of their own, deposit, dehydrate, store and leave in the
honey comb to
ripen and mature" (Revised Codex Standard for Honey, 2001).
Nectar typically includes approximately 14% simple sugars (w/w), 1% phenol
compounds,
and 85% water. The phenol compounds give the honey its taste, aroma and
colour. In the
warm conditions of the hive, typically 36 C, the nectar would very quickly
ferment. To
prevent this, the nectar is mixed with secretions, containing enzymes, from
the salivary and
hypopharyngeal glands of foraging bees. In the hive the nectar is passed from
bee to bee
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and more secretions are added before it is stored in the cells of the hive.
The amount of
enzymes present varies with the age, diet and physiological stage of the bees
(when a bee
is a forager its glands produce more digestive enzymes), strength of the
colony,
temperature of the hive, and the nectar flow and its sugar content.
5 The enzymes added to nectar by bees include diastase, which catalyses the
conversion of
starch to dextrin and sugar, Invertase, which catalyses the conversion of
sucrose to
fructose and glucose, and glucose oxidase, which catalyses the conversion of
glucose to
hydrogen peroxide and gluconic acid. Low doses of hydrogen peroxide prevent
the growth
of yeasts that would quickly ferment the nectar. As the bees progressively dry
the nectar to
10 form honey, the gluconic acid makes the honey acidic (between pH 3.5 and
4.5). Water is
effectively trapped to the sugar molecules in the honey and is not available
for further
chemical reactions. The amount of 'free' water in honey is measured as the
water activity
(aw). The range of aw found in honey has been reported to be 0.47-0.70, with
mean values
of 0.562 and 0.589 (RCIEGG, M; BLANC, B, 1981, The water activity of honey and
related
15 sugar solutions. Lebensmittel-Wissenschaft und Technologie 14: 1-6). The
aw of ripened
honey is too low to support the growth of any species, with no fermentation
occurring if the
water content is below 17.1% (MoIan, P. C. (1992). The antibacterial activity
of honey: 1.
The nature of the antibacterial activity. Bee World, 73(1), 5-28). The acidity
of the honey
and the lack of free water prevent the further risk of fermentation, and stop
the glucose
20 oxidase working. Honey also contains variable amounts of catalase
originating from the
nectar.
A typical chemical composition of blossom honey is:
Table 1. Typical honey composition
Component Blossom honey 1,
Min-Max
Average
(% w/w) (% wiw)
Water content 117,2 15 - 20
Fructose 38,2
Glucose 131,3 .. Tr24 - 40
Sucrose 0,7 .0,1 - 4,8
Other
disaccharides 5
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Total sugars 79,7 =
IMinerals 0,2 0,1 - 0,5 ----
lAmino acids,
1Proteins 10,3 10,2 - 0,8
Acids 0,5 0,2 -0,8
pH 3,9 3,5 - 4,5
In addition, trace amounts of pollen are present, which can be used to
identify the botanical
origin of the honey, as well as the enzymes invertase, diastase, catalase, and
glucose
oxidase. There is also phytochemical component. This varies but is typically
up to -1%,
depending on the source of the honey.
Once diluted, the glucose oxidase present in natural honey is able to convert
glucose
substrate in the diluted honey to release hydrogen peroxide. However, the
variability in the
content of honey (particularly in the content of glucose oxidase activity,
glucose, and
catalase activity) means that honeys from different sources, or different
harvests of honey
from the same source, can be very variable in their antimicrobial
effectiveness.
According to an embodiment of the invention, the honey may be pasteurised.
Pasteurisation of honey inactivates the catalase and glucose oxidase activity
present in the
honey. Optionally, the pasteurised honey may be filtered to remove any
particles (such as
wax particles and bee wings) that may be in the honey post-harvest. To form a
storage-
stable composition of the invention, a glucose oxidase is contacted with the
pasteurised
honey once it has cooled to a temperature (suitably 35-40 C) that will not
inactivate the
added glucose oxidase and at which the honey remains sufficiently liquid to
facilitate mixing
with glucose oxidase.
Honey can be pasteurised at a temperature that is sufficient for the heat
inactivation of
catalase activity. A suitable minimum temperature is from 6000 to 80 C. This
temperature
should be maintained preferably for at least two minutes.
The control of the heat process may be important, since a bi-product of
heating honey is
the formation of HMF (HydroxyMethylFurfuraldehyde) which is used as an
indicator of heat
and storage changes in honey. HMF is formed by the breakdown of fructose in
the
presence of acid. Heat increases the speed of this reaction. The increase in
speed is
exponential with increasing heat. For every degree that the honey is raised
above 40 C,
close to the normal hive ambient temperature, HMF increases rapidly. HMF is
not a harmful
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product. Jams, Molasses, Golden Syrup etc. can have levels of HMF 10 to 100
times that
of honey. However HMF levels are used as an indication of degradation of honey
and
under the Codex Alimentarius Standard 40 mg/I is the maximum permissible level
in the EU
for table honey.
To prevent the build up of HMF it is preferred that the honey is raised
rapidly to
temperature levels to inactivate the catalase and then the honey is brought
quickly down in
temperature to a maximum of between 40 and 45 C using a heat exchange
mechanism.
No water is added during the process of this preferred embodiment, and so the
resulting
composition does not include sufficient free water to allow the glucose
oxidase to convert
the glucose present to release hydrogen peroxide. The storage-stable
composition
comprises: pasteurised honey, and added glucose oxidase. There is no
detectable
hydrogen peroxide present. The composition can be stored at ambient
temperature for at
least several days.
In other embodiments of the invention, the honey may be unpasteurised.
According to some preferred embodiments, the honey (pasteurised or
unpasteurised) is a
creamed honey. Creamed honey is a honey that has been processed to control
crystallization. Creamed honey contains a large number of small crystals,
which prevent
the formation of larger crystals that can occur in unprocessed honey. A method
for
producing creamed honey was described in U.S. Patent 1,987,893. In this
process, raw
honey is first pasteurised, then previously processed creamed honey is added
to the
pasteurized honey to produce a mixture of 10% creamed honey and 90%
pasteurised
honey. The mixture is then allowed to rest at a controlled temperature of 14
C. This method
produces a batch of creamed honey in about one week. A seed batch can be made
by
allowing normal honey to crystallize and crushing the crystals to the desired
size. Large
scale producers have modified this process by using paddles to stir the honey
mixture
while holding the mixture at 14 C. In alternative creaming methods, the
pasteurisation step
may be omitted, with the honey instead being slowly warmed to 37 C.
In other embodiments of the invention, the honey (pasteurised or
unpasteurised) is an
uncreamed honey. For example, the honey may be a pasteurised, uncreamed honey.
The glucose oxidase may be a purified natural glucose oxidase preparation
which is of
medical grade or medical device grade for medical applications. The activity
of the glucose
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oxidase may be selected depending on the desired rate of production of
hydrogen peroxide
following dilution of the composition. Several glucose oxidase preparations
are
commercially available (glucose oxidase is identified by the reference
CAS:9001-37-0).
Common microbial sources for glucose oxidase from non genetically modified
organisms
include selected strains of Aspergillus niger, Penicillium amagasakiense,
Penicillium
variabile, Penicillium notatum. Medical device grade glucose oxidase, from GMO
Aspergillus niger, is available from Biozyme UK, activity 240iu/mg. Food
standard glucose
oxidase, from Aspergillus niger, is available from BIO-CAT INC, activity
15,000 Units/g.
Non-Genetically Modified glucose oxidase is available from BIO-CAT INC,
activity
12,000/g. Glucose oxidase (G03B2), from Apsergillus niger, is available from
BBI Enzymes
Limited, activity 360 Units/mg. Contaminants: alpha amylase no greater than
0.05%,
Saccharase no greater than 0.05%, maltase no greater than 0.05% and GO/Cat no
less
than 2000.
The enzyme activity (for example, the glucose oxidase activity) may range, for
example,
from 1-400 IU/mg, or 1-300 IU/mg, for example 250-280 ILI/mg. The amount of
enzyme
used is likely to depend on several factors, including the desired use of the
composition,
the amount of any catalase activity present in the substance, the amount of
substrate
present in the substance, the desired level of hydrogen peroxide release, and
the desired
length of time for hydrogen peroxide release. A suitable amount of enzyme can
readily be
determined by a person of ordinary skill in the art, if necessary using a well
diffusion assay,
to determine the extent of hydrogen peroxide release for different amounts of
enzyme.
Suitable amounts of enzyme (such as glucose oxidase) may be from 0.0001% to
0.5% w/w
of the composition. The amount of enzyme used may be selected so as to produce
a
composition for generating antimicrobial activity that is equivalent to a
selected phenol
standard (for example a 10%, 20%, or 30% phenol standard).
Compositions of the invention, particularly compositions in which the
substance is honey
(for example, unpasteurised honey), and the enzyme is glucose oxidase that is
able to
convert D-glucose in the honey to release hydrogen peroxide, may comprise at
least 1 unit,
and preferably up to 1500 units, of glucose oxidase per gram of the
composition. The
glucose oxidase is additional (i.e. added as a result of human intervention)
to any glucose
oxidase activity that may naturally be present in the substance.
A "unit" is defined herein as the amount of enzyme causing the oxidation of 1
micromole of
glucose (or other enzyme substrate) per minute at 25 degrees centigrade at pH
7Ø
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The Applicant has found that the antimicrobial potency of compositions of the
invention
may be increased simply by increasing the amount of glucose oxidase activity
present in
the composition.
In some embodiments of the invention, a composition of the invention comprises
more than
15 units, for example at least 30 units, at least 50 units, or at least 100
units, and suitably
less than 685 units, for example 100-500 units, of glucose oxidase per gram of
the
composition. Such compositions have been found to have superior antimicrobial
properties
than compositions with up to 15 units of glucose oxidase per gram of the
composition. In
particular, such compositions have increased potency against a wide range of
microorganisms, including MSSA, MRSA, Group A and B Streptococci,
Enterococcus,
Ecoli ESBL, Serrliquefaciens Amp C, Kleb.pneumoniae, Pseudomonas aeruginosa,
Acinetobacter baumannii, and Candida albicans.
In other embodiments of the invention, a composition of the invention
comprises at least
500 units, for example 500-1000 units, or 685-1000 units, of glucose oxidase
per gram of
the composition. Such compositions have been found to have even more superior
antimicrobial properties. In particular such compositions have further
increased potency
against a wide range of microorganisms, including Staphylococcus aureus, MSSA,
MRSA,
Group A and B Streptococci, Enterococcus, E.coli, Ecoli ESBL, Serrliquefaciens
Amp C,
Kleb.pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Candida
albicans.
Compositions of the invention can be used to treat any microbial infection
that can be
treated by hydrogen peroxide. Examples include infection caused by gram
positive
bacteria, gram negative bacteria, acid-fast bacteria, viruses, yeasts,
parasitic or pathogenic
micro-organisms or fungi. In particular, infections caused by the following
micro-organisms
may be treated: Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa,
Candida albicans, Pro pionibacterium acnes, Staphylococcus aureus,
Staphylococcus
epidermidis, Staphylococcus saprophytics, Beta haemolytic Streptococci Group A
or B,
Campylobacter coli, Campylobacter jejuni, Methicillin Resistant Staphylococcus
Aureus
(MRSA), Methicillin Sensitive Staphylococcus Aureus (MSSA), Botrytis cinerea,
Mycobacterium tuberculosis, Cryptosporidium, Plasmodium, and Toxoplasma.
The pasteurisation process inactivates any enzyme activity present in the
honey, and so
there is no variability in catalase and substrate conversion activity between
pasteurised
honeys from different sources, or between different harvests of honey from the
same
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source. The amount of substrate conversion activity can be controlled by
addition of a
purified glucose oxidase preparation with a defined amount and activity of the
enzyme.
Thus, the inherent variability in antimicrobial properties between different
types and
harvests of honey is considerably reduced, and the antimicrobial properties of
honeys with
5 low antimicrobial potency are improved.
Compositions of the invention may be administered at an appropriate frequency
determined by the subject or a healthcare provider. Suitably compositions of
the invention
may be administered at least every several days, for example every week, but
preferably
several times a day, every day, or every other day.
10 The amount of a composition of the invention administered will depend on
many factors,
such as the strength of the antimicrobial properties of the composition, and
on the age and
condition of the subject to be treated. However, for many applications it is
expected that
each administration comprises 0.1-100g, 0.5-100g, 1-100g, 2-100g, 5-100g, 0.1-
10g, 0.5-
10, or 1-10g of a composition of the invention.
15 According to preferred embodiments of the invention, a composition of
the invention is
sterile.
Compositions of the invention may be sterilised by any suitable means. The
Applicant has
found that compositions comprising glucose oxidase retain glucose oxidase
activity (and,
therefore, the ability to release hydrogen peroxide on dilution) following
sterilisation by
20 exposure to gamma irradiation. A suitable level of gamma irradiation is
10-70 kGy,
preferably 25-70 kGy, more preferably 35-70 kGy.
Compositions of the invention preferably have not been sterilized by
zonation, and do not
include ozone, or any components that have been subjected to sterilisation by
zonation. In
particular, compositions of the invention should not comprise ozonized honey
or zonated
25 oil.
Sterilised compositions of the invention that are stored away from exposure to
light are
expected to retain stability for at least six months. For example, such
compositions may be
packaged in high-density polyethylene/low-density polyethylene (HDPE/LDPE)
tubes or in
polyester-aluminium-polyethylene (PET/Al/PE) sachets.
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A composition of the invention is preferably a medical grade or medical device
grade
composition. Preferably the unrefined natural substance is a honey, suitably a
medical
grade or medical device grade honey.
Preferably a composition of the invention comprises a creamed honey, more
preferably a
creamed unpasteurised honey. Such compositions can readily be administered
topically
because the presence or number of large crystals has been minimised by the
creaming
process.
For compositions of the invention that comprise honey, it will be appreciated
that there may
be no need to use pasteurised honey in the composition if the composition is
sterilised. It
may instead be preferable to use unpasteurised honey (preferably creamed
honey) or other
unrefined natural substance. In some embodiments, compositions of the
invention
comprise unpasteurised honey, and added purified glucose oxidase.
Thus, a storage-stable composition of the invention may comprise unpasteurized
honey,
and added purified glucose oxidase that, in the presence of sufficient free
water, is able to
convert D-glucose in the honey to release hydrogen peroxide, wherein the
composition
does not include sufficient free water to allow the glucose oxidase to convert
the D-glucose.
Such compositions may comprise at least 1 unit, and for example up to 1500
units, of
glucose oxidase per gram of the composition. Suitably such compositions
comprise more
than 15 units of glucose oxidase per gram of the composition, for example at
least 100
units, or 100-500 units, of glucose oxidase per gram of the composition, or at
least 500
units, or 500-1000 units, of glucose oxidase per gram of the composition.
The honey of such compositions may comprise a creamed unpasteurized honey.
A composition of the invention may be a pharmaceutical composition comprising
a
pharmaceutically acceptable carrier, excipient, or diluent.
A composition of the invention may be in a form suitable for administration to
a human or
animal subject. Suitable forms include forms adapted for topical
administration. Forms
suitable for topical administration include a topical ointment, cream, lotion,
oil, liniment,
liquid, gel, or a dissolvable strip. If a storage-stable composition is used,
this may be diluted
by liquid present at the site of administration (for example, by saliva),
leading to release of
hydrogen peroxide at the administration site.
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37
A composition of the invention may be present with at least one suitable
antimicrobial or
immunostimulatory component, excipient or adjuvant, or any other suitable
component
where it is desired to provide ability to generate antimicrobial activity.
Preferably, however,
the compositions do not include any antibiotic.
A composition of the invention may comprise "Surgihoney". Surgihoney is
unpasteurised
honey with added purified glucose oxidase. Three different preparations of
Surgihoney
have been made with different antimicrobial potencies:
SH1 Surgihoney: unpasteurised honey with 0.1% (w/w) added glucose oxidase. The
enzyme used was food grade glucose oxidase, from Aspergillus niger, from BIO-
CAT, INC,
activity 15,000 Units/g. Sealed sachets of the SH1 Surgihoney were gamma
irradiated at a
target dose of 11/6-14.2 kGy.
SH2 Surgihoney: unpasteurised honey with 0.1% (w/w) added glucose oxidase. The
enzyme used was glucose oxidase (G03B2), from Aspergillus niger, from BBI
Enzymes
Limited, activity 274 Units/mg. Unit Definition: the amount of enzyme causing
the oxidation
of 1 micromole of glucose per minute at 25 degrees centigrade at pH 7Ø
Contaminants:
alpha amylase no greater than 0.05%, Saccharase no greater than 0.05%, maltase
no
greater than 0.05% and GO/Cat no less than 2000.
SH3 Surgihoney: unpasteurised honey with 0.25% (w/w) added glucose oxidase.
The
enzyme used was glucose oxidase (G03B2) from BBI Enzymes Limited, activity 274
Units/mg.
Thus, SH1 Surgihoney contains 15 units of glucose oxidase per gram of the
composition,
SH2 Surgihoney contains 274 units of glucose oxidase per gram of the
composition, and
SH3 Surgihoney contains 685 units of glucose oxidase per gram of the
composition.
In some embodiments, a composition of the invention (in particular, a
composition of the
invention that comprises honey and added glucose oxidase ¨ referred to below
as "Active
honey") is not, or does not comprise the following:
Pornyriade
Active honey 25%
White petrolatum
Light liquid paraffin
Talc
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38
Kaolin
Zinc oxide
In some embodiments, a composition of the invention (in particular, a
composition of the
invention that comprises honey and added glucose oxidase ¨ referred to below
as "Active
honey") is not, or does not comprise the following:
Up balm
Petrolatum 5594 50%
Microcrystalline Wax 9%
Cyclomethicone D5 31%
Active Honey 10%
In some embodiments, a composition of the invention (in particular, a
composition of the
invention that comprises honey and added glucose oxidase ¨ referred to below
as "Active
honey") is not, or does not comprise the following:
Cream
Honey 15%
Carbomer 2.63%
Dimethicone 0.13%
Disodium Lauryl Sulphosuccinate 0.05%
Disodium Edetate 0.13%
Glycerol 5.26%
Silica Colloidal Hydrated 0.33%
Poloxamer 0.26%
Sodium Hydroxide 0.41%
Purified Water 85.03%
In some embodiments, a composition of the invention does not comprise:
A cream formulation comprising Surgihoney SH1 was made with the following
ingredients:
beeswax (for the lipophilic phase);
soya lecithin (as an emulsifier);
water; and
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39
SH1 Surgihoney,
In some embodiments, a composition of the invention does not comprise an
emulsion
comprising the following ingredients:
log Surgihoney dissolved in 10m1 of glycerol;
10m1 of paraffin oil;
1m1 Polyglycerol polyricinoleate (PGPR).
In some embodiments, a method of the invention does not comprise the following
method
for preparation of a Surgihoney emulsion, as recited in Example 7:
10g Surgihoney was dissolved in 10m1 of glycerol. 10m1 of paraffin oil was
then added to a
Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry
attached. lml of PGPR (Polyglycerol polyricinoleate) was then added. The
rheometer was
then started under the following conditions; Shear rate 2000 its, Temperature
set at 37.5
C. After 2 minutes, 10m1 of Surgihoney-glycerol solution was added dropwise.
Once a total
of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier
to a
container.
Embodiments of the invention are now described, by way of example only, with
reference
to the accompanying drawings in which:
Figure 1 shows the results of an assay for the cytotoxic activity of
Surgihoney;
Figure 2A shows different hydrogen peroxide production rates for Surgihoney
SH1, SH2,
and SH3;
Figure 2B shows the relationship between phenol activity and maximum hydrogen
peroxide
activity in Surgihoney SH1, SH2, and SH3;
Figure 3 shows time kill curves for Surgihoney 1 (Si), Surgihoney 3 (S3), and
Medihoney
(MH) for different test organisms: (a) Staphylococcus aureus; (b) Methicillin-
resistant
Staphylococcus aureus (MRSA); (c) E.coli; (d) vancomycin resistant
enterococcus (VRE);
(e) Pseudomonas aeruginosa; (f) Klebsiella; (g) Eco/i ESBL; (h) Enterococcus
faecalis;
and
Figure 4 shows an optical microscopy images of reverse micelles in an emulsion
containing
Surgihoney.
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Example
Sur.ginoney Crearp Forinulatorl
A cream formulation comprising Surgihoney SH1 was made with the following
ingredients:
beeswax (for the lipophilic phase);
5 soya lecithin (as an emulsifier);
water; and
SH1 Surgihoney.
The cream remains stable for many years, as judged by its ability to produce
hydrogen
peroxide when contacted with water.
10 Example 2
Anti-viral activity of Sumihoney
SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus Type 1 or 2 (HSV 1
or HSV
2) in cell culture medium (a 50% mixture of honey and virus in cell culture
medium) and
then incubated for 1 hour at 37 C. A dilution series was then made from the
mixture, and
15 the dilutions were plated onto Vero cells. SH1 Surgihoney reduced the
titre of virus by 1
log. SH2 Surgihoney was virucidal for both HSV 1 and HSV 2 (>6Iog drop in
titre). The
experiment was repeatable.
Example 3
Ant-viral activity of Sumihoney
20 SH1 or SH2 Surgihoney was mixed with Herpes Simplex Virus (HSV) (50j,Lg
honey and
50jil virus) and incubated for 1 hour at 37 C. A dilution series (102, 10-3,
10-4, 10-5) was
then made from the mixture, and the dilutions were used in a plaque reduction
assay.
Controls with no honey, or with control honey were also performed. The number
of viral
plaques formed for each dilution was recorded. The results are shown in the
Table below,
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41
Table 2. Anti-viral effect of Sul:Ohm-ley
.............................. --,4 ................ Experiment 1
Experiment 2
...,....,õ
=
Honey_J?ilution _.,;ii well 1 well 2 well 3 well 1 well 2 well 3
_2 : * * 9
-3 1 1 1 5 0 0 0
SH1 ¨
-4
...................... -5
_......:
sH2 1 7.3.
' -4 il 0 0 1 0 i 0 1 0 0
i -2 100 ___ 95 ___ 88 108 . 128 106,
"--
Control -3 13 15 11 14 12 = 15
.
,
Honey 1 -4 ii 2 1 1 2 3 2 i 2 '
-5 0 .. 0 0 .. : .. 0 0 1 1
2 160 158 ... : 164
..õ, õõ
1 _____________________ - 111111 ¨ ::
No I -3 :MI ........................................... 28 22 18
, Honey! ... -4 ............ : _ .. . 6 4 :i .
1 :
The results show that SH1 and SH2 Surgihoney was strongly virucidal against
HSV in both
experiments.
Example 4
ce.Y.tPAP?.(ic! '' ?ctiviligSurgibglgY.,
SH1 or SH2 Surgihoney (501.1g honey diluted 10-2, 10-3, 10-4, 10-5) was
incubated on cells
for 2 days. The number of live cells, and the total number of cells was
counted (percentage
viability = live/total x 100). The results are shown in the table below, and
in Figure 1.
The results show that SH1 Surgihoney was cytotoxic at the 10-2 dilution, and
cytostatic at
the 10-3 and 10-4 dilutions, and that SH2 Surgihoney was cytostatic at the 10-
2, 10-3 and 10-4
dilutions. SH1 and SH2 Surgihoney were not cytotoxic or cytostatic at the 10-5
dilution.
It is concluded from the results in Examples 3 and 4 that Surgihoney can be
administered
at doses which are virucidal but not cytotoxic or cytostatic.
42
0
t..,
=
Table 3.. Cytotoxic activity of Surciihoney
--.1
=
Number of live cells Total number of
cells ,g Percentage viability ,
c.,.)
g
_______________________________________________________________________________
__ g _______________________
.6.
.6.
f 1
1 i oe
Condition Dilution ..t rep1 rep2 Ave
standard standard
deviation rep1 rep2 Ave repl rep2
Ave standard
deviation
: ........ deviation
DMEM - % 1300000 2100000 1700000
565685.4 1400000 2600000 2000000 848528.1 92.9 80,8 86.8 8.5
-2 1200000 880000
0-1040000 226274.2 1300000 1000000 1150000
212132 92.3 88.0 90.2 3.0 4,
,
* i= ......... ,
Control a ',, 2700000 2400000 2550000 212132
,
...............................................................................
..................... 2800000 2600000 2700000 141421.4 96.4 92.3 94.4 2,9
r
...............................................................................
.............................
1 honey -4 3400000 2800000 3100000
424264.1 = 3600000 3000000 3300000 424264.1 94.4 93.3
93.9 0.8
,
............................... ,
.......................................................................... i
;
-5 _.......t 2100000 1300000 1 1700000 565685.4 2200000
1500000 1850000 494974.7 i 95.5 86.7 i 91.1 6,2
l -2 ,% 120000 70000
.............. 1 .............. 95000 35355.34
_...
_______________________________________________________________________________
___ 370000 350000 360000 14142.14 32.4
20.0 1 26.2
8.8
_______________________________________________________________________________
________________ _....._...., .. .............................. P
-3 j 380000 380000 .. 380000 0 400000 600000
500000 141421.4 95.0 63.3 1 79.2 22.4 = .
SH1 .......... ,5 -------------------- - 1
.............................. , ................................
-4 430000 780000 605000 247487.4 ,, 470000 850000
660000 268700.6 91.5 91.8 I 91.6 0.2 .
,
...............................................................................
................ .; ------------------ r.,
-5 1 1800000 2200000 1 2000000 282842.7
2000000 2400000 2200000 282842.7 90.0 91.7 90.8 1.2
..,
"
,
' -2 320000 360000 1 340000 28284.27 1
390000 400000 395000 7071.068 82.1 90.0 86.0 5.6
-1,- -------
......................................... i
a 450000 570000 510000 84852.81 ,,,' 760000 730000
745000 21213.2 59.2 78.1 68.6 13.3
SH2" 1
_______________________________________________________ i
; 4 460000 690000 575000 162634.6 660000 790000
725000 91923.88 69.7 87.3 78.5 12.5
__________________________________________________ ,
.
, _.
.......................... -1 . . f t "I
; -5 1600000 1700000 1650000 70710.68
1800000 2000000 1900000 141421.4 88.9 i 85.0 1 86.9 I 2.7 =
IV
n
,-i
rt
w
=
c7,
u,
w
w
u,
oe
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43
Example 5
Antimicrobial activity of Surgihoney.
The antimicrobial activity of Surgihoney (SH) and two prototype modified
honeys made by
Apis mellifera (honeybee) against Staphylococcus aureus (NCIMB 9518) was
tested. We
also examined a number of modified types of Surgihoney for the ability to
change the level
of production of hydrogen peroxide from the samples.
Methods: Surgihoney (SH) was compared with two modified honeys, Prototype 1
(PT1) and
Prototype 2 (PT2) using a bioassay method against a standard strain of
Staphylococcus
aureus. Further work studied the rate of generation of hydrogen peroxide from
these
preparations.
Results: Surgihoney antimicrobial activity was shown to be largely due to
hydrogen
peroxide production. By modification of Surgihoney, two more potent honey
prototypes
were shown to generate between a two- and three-fold greater antibacterial
activity and up
to ten times greater peroxide activity.
Conclusions: Surgihoney shows good antimicrobial activity. Two further honey
prototypes
have been shown to have antimicrobial activity that is possible to be enhanced
due to
demonstrated increases in peroxide activity.
Methods
1. Determination of Honey Activity by Bioassay Method
The antibacterial activity of Surgihoney (S) and two modified honeys,
Prototype 1 (PT1)
and Prototype 2 (PT2) was measured using Staphylococcus aureus (NCIMB 9518)
and
expressed as the equivalent percentage phenol. Values were calculated of the
mean from
three sample replicates tested, repeated on three days.
Assay Method. The agar well diffusion method used was adapted from the punch
plate
assay for inhibitory substances described in the Microbiology Standard Methods
Manual for
the New Zealand Dairy Industry (1982) [Bee Products Standards Council:
Proposed
standard for measuring the non-peroxide activity of honey. In. New Zealand:
Bee Products
Standards Council; 1982.].
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44
lnoculum Preparation. Overnight culture was adjusted to an absorbance of 0.5
measured
at 540 nm using sterile nutrient broth as a blank and a diluents and a cuvette
with a 1 cm
pathway.
Assay Plate preparation. A volume of 100 pi of the culture adjusted to 0.5
absorbance
__ was used to seed 150 ml nutrient agar to make the assay plates. The agar
was swirled to
mix thoroughly and poured into large petri dishes which had been placed on a
level
surface. As soon as the agar was set the plates were placed upside down
overnight before
using the next day. For assay these seeded plates were removed from 4 C and
allowed to
stand at room temperature for 15 min before cutting 7.0 mm diameter wells into
the surface
__ of the agar. 250 pl of test material (sample or standard) was placed into
each well.
Catalase solution. A 200 mg/ml solution of catalase from bovine liver (Sigma
C9322, 2900
units/mg) in distilled water was prepared fresh each day.
Sample preparation. Primary sample solutions were prepared by adding 4 g of
sample to
4 ml of distilled water in universals and placed at 37 C for 30 minutes to aid
mixing. To
__ prepare secondary solutions, 2 ml of the primary sample solution was added
to 2 ml of
distilled water in universals and mixed for total activity testing and 2 ml of
the primary
sample solution was added to 2 ml of catalase solution and mixed for non-
peroxide activity.
Preparation of phenol standards. Standards (w/v) 10%, 30%, 50% phenol were
prepared
by dissolving phenol in water. Phenol standards were brought to room
temperature in the
__ dark before use and were mixed thoroughly before addition to test wells.
Each standard
was placed in three wells to test in triplicate. Standards were kept at 4 C
with an expiry
date of one month.
Sample and standard application. All samples and standards were tested in
triplicate by
adding 250 pl to each of 3 wells.
__ Plate incubation. After application of samples the plates were incubated
for approximately
18 hours at 37 C. The diameter of inhibition zones, including the diameter of
the well (7.0
mm), was recorded.
Calculation of antibacterial activity of samples. The mean diameter of the
clear zone
around each phenol standard was calculated and squared. A standard graph was
plotted of
__ % phenol against the square of the mean diameter of the clear zone. A best-
fit straight line
was obtained using linear regression and the equation of this line was used to
calculate the
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activity of each diluted honey sample from the square of the mean measurement
of the
diameter of the clear zone. To allow for the dilution (assuming the density of
the
Surgihoney to be 1.35 g/m1) this figure was multiplied by a factor of 4.69 and
the activity of
the samples was then expressed as the equivalent phenol concentration (% w/v).
5 Total Activity: all the activity, including activity due to hydrogen
peroxide (H202).
Non-Peroxide Activity: H202 is removed by treating samples with catalase
enzyme.
2. Determination of Honey Activity by HA Method
The activity was measured using the Merckoquant0 1.10011. & 1.10081,
Peroxide Test Kits. Concentrations expressed as the equivalent mg/L H202
10 Samples were diluted 1:10 with purified water. Following 5 min
incubation, all samples
were measured for H202 production each hour over a 12 hour period followed by
24 and 48
hour time points.
Method of Determination. Peroxidase transfers oxygen from the peroxide to an
organic
redox indicator, which is then converted to a blue coloured oxidation product.
The peroxide
15 concentration is measured semi-quantitatively by visual comparison of
the reaction zone of
the test strip with the fields of a colour scale. The reaction zone of the
test strip is immersed
into the Surgihoney sample for 1 sec, allowing excess liquid to run off the
strip onto an
absorbent paper towel and after 15 seconds (Cat. No. 110011), 5 seconds (Cat.
No.
110081), after which a determination of the colour formed in the reaction zone
more
20 precisely coincided with the colour fields scale.
Results
1, Activity Rating
The antimicrobial activity produced by the modification of the honey samples
resulted in a
two-fold and almost three-fold respectively increase in phenol activity with
PT1 and PT2
25 compared with Surgihoney alone. The results for the three samples of
Surgihoney (SH)
and two modified prototypes, PT1 and PT2 are shown in the Table below.
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46
Table 4. The perpxide and non-peroxide antibacterial activities of Surgihoney
(SH) and two
modified prototypes. PT1 and P12 against Staphylococcus aurous (NCNB 9518),
Sample Name Batch No. Total Non-Peroxide
Activity (% Activity (1)/0 phenol)
phenol)
Surgihoney 2015-06-018B 32 0
Surgihoney PT1 HHI4110311 65 7
Surgihoney PT2 HHI14110312 83 10
2. Determination of Honey AOtiOty by HA Method
The prototype modifications are observed to generate up to seven and ten times
the
hydrogen peroxide activity of Surgihoney. The results for the three samples
are shown in
Figure 2A. By taking the maximum level of hydrogen peroxide output for each of
the three
honey prototypes and plotting this against the total phenol activity a linear
relationship is
observed (Figure 2B).
Discussion
The results from this work show that the main antimicrobial activity of
Surgihoney and two
modified prototypes, PT1 and PT2 are due to hydrogen peroxide. This is a
similar finding to
certain other honeys from a variety of floral sources. However, unlike
previous work the
availability of hydrogen peroxide from the samples is able to be enhanced and
at 12 hours
is seven and ten times respectively the value for Surgihoney alone. There is a
striking
linear relationship between the antimicrobial activity and the maximum output
of hydrogen
peroxide from the three honey prototypes.
This peroxide activity offers potent antimicrobial activity that is ideally
suited to treat or
prevent microbial infections. Hydrogen peroxide is an effective antimicrobial
and is already
used as a biocide for its potent activity against vegetative bacteria, yeasts
and spores. It
produces its antimicrobial effect through chemical oxidation of cellular
components.
The human toxicity of hydrogen peroxide is concentration dependent and one
study has
claimed that the differential concentrations for antimicrobial and human
toxicity might
overlap. By contrast, certain preparations of honey have been shown to be an
effective
antimicrobial agent by supplying low concentrations of hydrogen peroxide
continuously
over time rather than as a large amount and without such toxicity. Indeed
there is
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47
compelling evidence that where physiological levels of hydrogen peroxide are
applied to
mammalian cells there is a stimulation of biological responses and activation
of specific
biochemical pathways in these cells.
Clearly Surgihoney and the two modified prototypes, PT1 and PT2 offer
effective hydrogen
peroxide release over at least 24 hours.
Conclusions
Surgihoney and the two modified prototypes, PT1 and PT2 have been shown to
have
potent antimicrobial activity against a standard strain of Staphylococcus
aureus. These
antimicrobial activities have been shown to be due to hydrogen peroxide. The
activity is
scalable and can be described in terms of hydrogen peroxide activity. These
modified
honeys are effective, non-toxic and easy to administer.
Example 6
in vitro antimicrobial activity of Suraihoney
This example describes susceptibility testing of a range of bacterial isolates
to Surgihoney
by disc diffusion method, minimum inhibitory concentration (MIC) and minimum
cidal
concentration (MBC) determination, and time bactericidal measurements.
Summary
Results: Surgihoney demonstrates highly potent inhibitory and cidal activity
against a wide
range of Gram positive and Gram negative bacteria and fungi. MIC/MBC's are
significantly
lower than concentrations likely to be achieved in topical clinical use.
Surgihoney 1
MIC/MBC's for Staph. Aureus are 31 and 125gms/L and Surgihoney 3 MIC/MBC's
0.12
and 0.24gms/L.
Cidal speed depends on the potency. In Surgihoney 1, the least potent,
complete cidal
activity occurs for all organisms tested within 48 hours. For Surgihoney 3,
the most potent,
cidal activity occurs within 30 minutes. Maintenance of the Surgihoney
inoculums
preparation for up to a week demonstrated complete cidal activity and no
bacterial
persistence.
Conclusions: Surgihoney has wide potential as a highly active topical
treatment combining
the effects of the healing properties of honey with the potent antimicrobial
activity of the
bioengineered product. It is highly active against multidrug resistant
bacteria. It is more
active than other honeys tested and comparable to chemical antiseptics in
antimicrobial
activity.
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This study examines the in-vitro properties of Surgihoney. Surgihoney retains
all the
established healing properties of natural honey but its antimicrobial activity
can be set at
whichever potency is required. This study determined minimum inhibitory
concentrations
(MIC) and minimum bactericidal concentrations (MBC) of Surgihoney 1, 2 and 3
and time
kill curves.
Methods
Surgihoney was provided as potency grades 1, 2 and 3. It was presented as a
sterile
pharmaceutical grade product in a sachet in semisolid form.
Clinical isolates were collected from soft tissue microbiology samples.
Eighteen isolates of
Staphylococcus aureus, 12 methicillin-sensitive (MSSA) and 6 methicillin-
resistant (MRSA),
6 isolates of13 haemolytic streptococci, Lancefield groups A (2), B (2), C
(1), G (1), 5
isolates of Enterococcus spp. Including vancomycin-resistant E. faecium, 6 of
Esch. coli,
including extended spectrum 13 lactamase producers, 2 of Klebsiella spp., 1
Serratia
Marcescens Amp C producer, 4 of Pseudomonas aeruginosa, 1 of Acinetobacter
Iwoffii, 1
of Propionibacterium acnes, 1 Bacteroides fragilis, and 2 of Candida albicans,
1 of Candida
glabrata, 1 of Aspergillus fumigates were tested against Surgihoney.
Agar diffusion
Six mm wells were cut in isosenitest agar which had already been inoculated
with the test
organism at a concentration to give a semiconfluent growth. Test Surgihoney
and other
honeys in the pilot study were added to the wells.
A pilot study was carried out initially to compare Surgihoney potencies Si,
S2, S3 with a
variety of honeys from around the world, European, South American, New
Zealand,
Yemani, Sudanese and with medical honey, Medihoney and with antimicrobial
dressings
containing silver (Silver Aquacell) and iodine (lodoflex). Wells were cut in
the plates
inoculated with Staphylococcus aureus and filled with test honey or in the
case of the
dressings, these were cut to 2x2cm and placed on the surface of the inoculated
plates.
Following the pilot studies the Surgihoney potencies Si, S2, S3 were tested
alone against
the range of bacterial isolates from skin lesions. The wells were filled to
the surface with a
preparation of approximately 2gms neat Surgihoney of the three potencies,
diluted and
emulsified in an equal volume of sterile water. Zone sizes were measured after
18-24 hours
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49
aerobic incubation (longer for Candida and Aspergillus spp., and anaerobically
for
Propionibacterium sp. And Bacteroides sp.)
Minimum Inhibitory Concentrations and Minimum Bactericidal Concentrations
Surgihoney product was warmed to 37 C to liquefy it and 5gms was mixed with
10mL
sterile deionised water. This dilution was regarded as the 'neat' substance
for serial
dilution. The British Society of Antimicrobial Chemotherapy (BSAC) method for
performing
minimum inhibitory concentrations (MIC's) and minimum bactericidal
concentrations
(MBC's) was used (Andrews JM. Determination of minimum inhibitory
concentrations. ..1
Antimicrob
372 Chemother 2001; 48(Supp 1): 5-16). The Surgihoney products were serially
diluted in
microtitre tray wells from neat to 1 in 1024. 75pL of each honey dilution was
added to each
well in the strip of the microtitre tray. The neat concentration represented a
concentration of
250gm/L and the 1 in 2048 dilution, approximately 0.12gm/L.
The test organisms were prepared by taking four morphologically identical
colonies for
each organism from pure culture to create a 0.5 McFarland density. This was
further diluted
1:10.
All wells including controls were inoculated with 75pL of the test isolate
preparation. The
well trays were incubated at 37 C for 18 hours. The M1C was regarded as the
most dilute
well that showed no detectable turbidity.
The MIC well and those around the M1C well were sub-cultured on blood agar and
incubated at 37 C for 18 hours to determine the MBC. The MBC was the most
dilute
concentration which showed no growth after incubation.
Time kill curves
The test organism inoculums was prepared by taking 0.1mL of a 0.5 MacFarlane
density of
the test organism and inoculating this in 3mL of nutrient broth. The test
inoculums was
divided into 3 separate bijous, a control and three test preparations to which
were added
0.5g of Surgihoney 1 (Si), Surgihoney 3 (S3) or Medihoney (MH). Colony counts
of the
inocula were determined by serial dilution 1:10 and plating 0.1mL on a blood
agar plate,
repeated 3 times.
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The test and control inocula were kept at 30 C to simulate the temperature of
a superficial
skin lesion. Colony counts were performed as above in triplicate at time 0.5,
2, 4, 24, 48, 72
and 168 hours.
5 A terminal culture was performed by inoculating 0.1m1 of the original
inoculums into nutrient
broth to neutralise any residual effect of the Surgihoney and incubating for
72 hours at
37 C, before plating on blood agar to determine test organism survival.
Results
10 Inhibitory zone sizes.
The pilot comparative studies demonstrated that all the Surgihoney potencies
had greater
antimicrobial activity than any other honey tested including the medical grade
honey,
Medihoney. The inhibitory zones for S1 were larger than those produced by any
other
honey. Silver dressings produced some inhibitory effect beneath the dressing
but there was
15 no zone of inhibition as there was for Surgihoney. Iodine dressings
produced a large zone
of inhibition (approximately 70mm) to Staphylococcus aureus , larger than S1
(36mm) and
equivalent to S3 (67mm).
In the quantitative zone size testing, Surgihoney at all potencies produced an
inhibitory
20 zone in agar diffusion against all bacteria tested, both Gram positive
and Gram negative
bacteria including multiply antibiotic resistant bacteria, and fungal species.
The zone size
for each species increased with increasing Surgihoney potency preparations.
Table 5. The
inhibitory effect of Surgihoney was not dependant only on direct contact with
the active
agent as with the silver dressings, but diffused well beyond the well
producing the
25 extensive zones listed in Table 5.
MIC's & MBC's
Surgihoney demonstrated significant antimicrobial activity against all the
isolates tested.
MIC's and MBC's were very consistent amongst isolates of the same species
whether the
30 isolates were multidrug resistant or highly sensitive. Table 6 lists the
MIC and MBC values
for isolate species tested by dilution ratio and Table 7 shows the MIC and
MBC's in grams
per litre. The degree of potency rose with the grade of Surgihoney. The MBC
for each
isolate was close to the MIC within a single dilution in most cases.
35 Topical concentration of Surgihoney is estimated at approximately
500gms/L. Surgihoney 1
MIC/MBC's for Staph. Aureus are 31 and 125gms/L and Surgihoney 3 MIC/MBC's
0.12
and 0.24gms/L respectively.
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Time kill curves.
Surgihoney kills bacteria rapidly. Starting with a colony forming units per
millilitre (cfu/mL)
of approximately 105, cfu/mL numbers in the control rose steadily, whereas in
the
Surgihoney inocula the cfu/mL fell rapidly after contact with both potencies
of Surgihoney.
By 30 minutes cfu numbers had fallen 1000 fold in most cases for both S1 and
S3 (Figure
3). For Si bacterial growth was undetectable by 2 hours in most cases and for
S3 by 30
minutes. Enterococci appeared more resilient and persisted for 48 hours. Cidal
activity was
complete for all organisms as terminal culture in nutrient broth with
subsequent plating on
blood agar failed to detect any organism in the S1 or S3 inocula.
Discussion
Surgihoney is natural honey which is also organic in the current sense of the
word in that it
has no agricultural additives or antimicrobial residues unlike much commercial
honey for
human consumption. It is not dependant on particular nectar sources, unlike
honeys such
as manuka which depends on a specific plant nectar source for its enhanced
activity. The
antimicrobial activity can be controlled in Surgihoney by the preparation
process allowing
the production of different grades with measured potency which is consistent.
This study has clearly demonstrated the efficacy of Surgihoney as a highly
potent
antimicrobial, active against all species of bacteria and fungi tested. In the
preliminary pilot
studies comparing Surgihoney with a variety of honeys sourced from around the
world and
with medical grade honey, Medihoney, Surgihoney demonstrated significantly
greater
antimicrobial efficacy. By comparison with the commonly used topical
antiseptics silver and
iodine, Surgihoney 3 produced an antimicrobial effect as great as iodine
dressings and
greater than silver dressings (Aquacel Ag) which was only effective at
inhibiting bacteria in
direct contact with the dressing.
M1C and MBC testing show that Surgihoney not only inhibits but also kills
microbes at
concentrations 10 to a 1000 fold below those that are likely to be achieved in
topical
treatment, estimated at 500gms/L. The cidal activity of Surgihoney occurs at
concentrations
close to its inhibitory activity. There is therefore the potential for
Surgihoney to be highly
active in polymicrobial inhibition and eradication when applied topically.
The speed of cidal activity is shown by the time kill curves to be extremely
rapid, within 30
minutes for Surgihoney 3 and within 2 hours for Surgihoney 1. This is the case
for both
Gram-positive and Gram-negative organisms, although enterococci appear
slightly more
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resilient. Fungi, Candida spp. Aspergillus sp. also require higher
concentrations and more
prolonged exposure to inhibit growth and kill the organism.
These in vitro studies have demonstrated the potential of Surgihoney with high
antimicrobial activity whose potency can be controlled.
Conclusion
These in vitro results support the clinical use of Surgihoney as a potent and
non-toxic
antimicrobial.
Table 5 Inhibitory zones sizes with diffOrept potencies of Surqihoney (S1, S2,
S3).
Bacteria No. of S1 Mean zone S2 Mean zone S3 Mean-zone¨
strains (range)/mm (range)/mm
(range)/mm
Methicillin-sensitive 12
36.2 (32-38) 53.4 (44-58)
66.5 (60-72)
Staphylococcus
aureus (MSSA)
Methicillin-resistant 6 35.6 (31-38) 52.6 (48-59)
67.3 (59-73)
Staphylococcus
aureus (MRSA)
Streptococci Beta 6 40.0(35-42) 44.5(38-51)
59.2 (53-69)
haemolytic
Enterococcus spp 5 38.0 (34-39) 49.5 (44-55)
61.8 (59-64)
Escherichia coli 6
33.4 (30-37) , 49.5(36-55)
62.7 (59-69)
Klebsiella sp. 2 3iL2 (30-3a) 40.0 (38-42)
57.0 (52-62)
Pseudomonas 4 25.8 (20-28) 34.8 (30-38)
50.2 (46-51)
aeruginosa
Acinetobacter Iwoffil 1
Bacteroides fragilis 1 : 22.3 28.7 34.2
Propionibacterium 1 19.7 23.4 31.9
acnes
Candida sp. 2 9(8-10) 15(15) 26
(24-28)
Aspergillus 1 8 12 18
fumigatus
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Table 6. Serial double dilutions from neat SurgponeifS1, S2,S31.showinq
dilution of
minimum inhibitory concentration (WC) and minimum bactericidal concentration
(MBC).
.............. T ...................................
S1 S2 ... I S3
.................................................... 1----
Organism name MIC MBC MIC MBC MIC MBC
MSSA 1:8 1:2 1:32 1:16 1:2048 1:1024
'. MRSA 1:16 1:4 ' 1:32 I. 1:16 1:2048
1:1024 .
Group B 1:64 1:16 1:64 1:64 1:1024 1:256
Streptococci
Group A 1:32 1:16 1:128 1:64 1:1024 1:512
Streptococci
Enterococcus 1:8 1:2 1:32 1:4 1:256 1:64
E.coli 1:8 1:4 1:64 1:64 1 1:256 1:128
Ecoli ESBL 1:8 1:2 1:64 1:64 1:256 j
1128
:. ......
Serrliquefaciens 1:8 1:4 1:16 1:4 1:256 1:128
Amp C
Kleb.pneumoniae 1:4 1:2 1:32 1:32 1:256 1:128
' Pseud.aeruginosa 1:16 1:16 1:64 1:16 1:256 1:64
Candida albicans 1Turbidat Growth at 1:16 1 1:16 1:64
1:64
neat neat
Table 7. Surdihoney MIC and MBC values expressed in 'Grams/Litre
........................................................................ ,
S1 S2 S3
Organism name MIC MBCr=-= ...............
MIC $ MBC MIC MBC
i. .........................................................
MSSA 31 125 7.8 .. 1 .. 15.6 0.12 0.24
L.....-.. . ................................. t=
: MRSA 15.6 62.5 7.8 15.6 0.12 0.24
:
Group B 3.9 15.6 3.9 3.9 0.24 0.9
Streptococci4. .........................................................
.
:
:
Group A 1 7.8 15.6 1.9 3.9 0.24 0.48
, Streptococci i
i Enterococcus I 31 125 7.8 ..... i 62.5 0.9 3.9
i ................................
i. ............
I E.coli 31 62.5 3.9 3.9 0.9 1.9
E.coli ESBL l' 31 : 125 3.9 3.9 0.9 1.9
Serrliquefaciens T. 31 : 62.5 15.6 62.5 0.9 1.9
Amp C
......................... 4, ....
Kleb.pneumoniae 1:4 125 7.8 7.8 0.9
: ..............................
Pseud.aeruginosa i 15.6 15.6 3.9 15.6 0.9 3.9 1
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Candida albicans Turbid at Growth at 156 15 6 s.9 : 3 9
'
neat 1 neat
Example 7
Surgihoney emulsion
Prew ration
10g Surgihoney was dissolved in 10m1 of glycerol. 10m1 of paraffin oil was
then added to a
Rheometer (TA Instruments AR-G2) which had a Jacket Peltier and vane geometry
attached. lml of PGPR (Polyglycerol polyricinoleate) was then added. The
rheometer was
then started under the following conditions; Shear rate 2000 its, Temperature
set at 37.5
C. After 2 minutes, 10m1 of Surgihoney-glycerol solution was added dropwise.
Once a total
of 10 minutes had elapsed the emulsion was transferred from the Jacket Peltier
to a
container.
Optical Microscopy
Optical microscopy revealed that the emulsion contained reverse micelles which
encapsulated Surgihoney. Such micelles can be observed in Figure 4. The
average micelle
diameter was found to be 178 pm.
Hydrogen peroxide tests
Hydrogen peroxide stick tests (Purchased from Sigma Aldrich (Quantofix0)) were
used to
detect hydrogen peroxide in the emulsion. The tests were carried out before
and after
addition of water, and showed that before addition of water, the emulsion
produced no
hydrogen peroxide, and after water was added, the emulsion tested positive for
hydrogen
peroxide. A positive test was indicated by a colour change to blue.
Stability Test
The emulsion maintained its capacity to generate hydrogen peroxide after
storage at
ambient conditions for at least four weeks.
$prAy jggl
The emulsion was added to a pump-action spray bottle and was found to be
sprayable.
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Example 8
Effects of different parameters on stability of Suroihoney emulsions
The effects of changing the Surgihoney emulsion preparation method described
in
Example 7, one parameter at a time, were investigated. The changes and their
effects are
5 summarised below.
i) PrOtiOttiOti.Ofthk bittlhase to the Surgihoney-cilyceroi phase
Oil volumes greater than 10m1, and less than 10m1, were tested. The emulsion
was found
to be more stable when a lower volume of oil was used compared to the volume
of the
Surgihoney-glycerol phase. When the volume of the oil was less than 6m1, the
emulsion
10 was found to separate by less than 3% in total volume over 72 hours. A
volume of 4m1
allowed a separation of just 1.3% of the total volume over 72 hours. This
stability is far
greater than that of the method described in Example 7, which provided an
emulsion with a
separation of 9.4% of total volume over the same time period.
yoluniqi.Of Pppfl
15 PGPR volumes up to 4m1, and less than 1m1, were tested. The emulsion was
more stable
when a higher amount of PGPR was used. At a volume of 4m1, PGPR provided
greater
stability than with use of lower volumes, and far greater stability than that
of the emulsion
described in Example 7.
20 iii) Shear rate::
Shear rates from 1000 1/s to 3000 1/s were tested. The emulsion was more
stable when a
higher shear rate was applied. A shear rate of 3000 1/s produced the most
stable emulsion.
Separation of only 4.6% of the total volume over 72 hours was observed for
emulsion
prepared at this shear rate, compared with a separation volume of 9.4% of the
total volume
25 over the same time period for emulsion prepared as described in Example
7.
iv) Temperature
Temperatures from 20 C to 40 C were tested. There was no noticeable trend
regard the
stability of the emulsions as temperature was increased. However a temperature
of 40 C
produced the most stable emulsion. Separation of only 3.1% of the total volume
over 72
30 hours was observed for this emulsion.
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v) Lendth of shear
Shear times of 20 minutes and 30 minutes were tested, in addition to that used
in the
preparation method described in Example 7. However, there was no significant
difference
produced by extending the shear time.
vi) Order oligacLant asIglitipp
The effect of changing the order in which the reagents are added to the
rheometer was
tested. The effect of adding all of the components before starting the
rheometer was
compared with the effect of adding the Surgihoney-glycerol and oil components
first, then
adding the PGPR after 1-2 minutes. The most stable emulsion was formed when
the PGPR
was added last. The resulting emulsion provided a separation volume of 2.8% of
the total
volume over 120 hours.
vii) Concentration of Surgihoneidissolved in _glycerol
The following ratios of Surgihoney (g) to glycerol (m1) were tested: lg:lml;
0.5g:1m1;
2g:lml. The ratio that produced the most stable emulsion was 1g:lml, the same
ratio used
in the preparation method described in Example 7.
viii) Sodium chloride
When sodium chloride is dissolved in the polar layer of the emulsion, it
increases the
polarity of this layer. It also forms electrostatic interactions with the
lipid layer of the
emulsion. The electrostatic interactions and increased polarity could improve
stability and
reduce coalescence. However, addition of sodium chloride (1g, 2g or 4g) was
not found to
influence the stability of the emulsion.
The effects of the changes are summarised in the table below:
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Table 8
Emulsion SH Glycerol ParaffinTPGPR Shear Temp. Order of
Stability
(0) (ml) Oil (ml) (ml) rate (1/s) ( C)
addition (% total vol.
after 72 hrs)
'6 ' 10 10 10 1 2000 37.5 Oil, then 9.4
PGPR, then
SH/glycerol
õ ...........
Ex a "(1) 10 10 6 1 1600 37.5 Oil, then 2.8
PGPR, then
SH/glycerol
EX 8 (i) .10 10 4 1 2000 37.5 Oil, then 1.3
PGPR, then
SH/glycerol
Ex 8 (ii) 10 10 µ70-7-11- ' 2000 37.5 - Oil, then 2.7
PGPR, then
SH/glycerol
Ex 8 (iii) 10 10 10 H 3000 37.5 Oil, then 4.6
PGPR, then
SH/glycerol
Ex 8 (iv) 10 10 10j -0-6- 40.0 Oil, then 3.1
PGPR, then
SH/glycerol
Ex 8 (vi) 10 10 10 1 "2000 37.5 SH/glycerol 2.8*
and oil, then
PGPR
* (after 120 hrs)
Example 9
Surgihoopy ejnylsi911$.Wittl hiCitt'stability
¨
The results from the changes described in Example 8 were used to design a
further
method of preparing Surgihoney emulsion. This method is described below.
PreOration
10g Surgihoney was dissolved in 10m1 of glycerol. 4, 6, 8, or 10m1 of Paraffin
oil was then
added to the rheometer (TA Instruments AR-G2) which had a Jacket Peltier and
vane geometry
attached. 10m1 of Surgihoney-glycerol solution was then added to the
rheometer. The
rheometer was then started under the following conditions; Shear rate 3000 us,
Temperature
40 C, gap 4000pm, Run time 10 minutes. After 1 minute 4m1 of PGPR
(Polyglycerol
polyricinoleate) was then added. Once a total of 10 minutes had elapsed the
emulsion was
transferred from the rheometer to a container.
Table 9
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Formulation Surgihoney- PGPR (ml) Ti Paraffin oil Total
Volume Total 1
number Glycerol (ratio - (ml) Separation Volume
1g1m1) (m1) <11 Days
Separation
( /0) after 20
........ 1 10 4 10 ________ 0 0.7
__
2 10 ........ 4 4 0 09
..
3 10 4 4 0 .................. 1.0
4
4 4 ........ 0 1.6
5
10
1.2
6
10 4 8 L 0 0.9
All of the formulations were found to be highly stable, with a slight increase
in stability observed
as the volume of paraffin oil used was increased.
5 Example 10
Surgihoney Cream Formulation
1.5g of Surgihoney was dissolved in 1.5ml of glycerol. lg of sodium alginate
was then dissolved
in the Surgihoney-glycerol solution. Next 10m1 of Paraffin oil was added to
the Rheometer (TA
Instruments AR-G2) which had a Jacket Peltier and vane geometry attached. lml
of PGPR
10
(Polyglycerol polyricinoleate) was then added. The rheometer was then started
under the
following conditions; Shear rate 2000 its, Temperature set at 37.5 C, gap
4000pm, Run time
10 minutes. After 2 minutes, 1.5ml of the Surgihoney-alginate and glycerol
solution was added
to the rheometer. After 3 minutes 8m1 of calcium chloride solution was added
dropwise to the
rheometer. Once a total of 10 minutes had elapsed the emulsion was transferred
from the
Jacket Peltier to a container.
Example 11
Non-aatteous Surciihoney Cream Formulation
The method described in Example 10 uses water to dissociate calcium chloride
into its ions.
This could potentially activate the Surgihoney to produce hydrogen peroxide,
and limit the
stability of the cream formulation. However, we have appreciated that calcium
chloride can be
dissociated using non-aqueous solvents, such as ethanol or acetic acid. We
have also
appreciated that glycerol is able to bind to free water. This property allows
water to be used to
dissolve the alginate, provided sufficient glycerol is present to prevent
premature release of
hydrogen peroxide.
The method described below uses ethanol as a solvent for calcium chloride, and
glycerol to
bind free water in the alginate solution.
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lg of sodium alginate is dissolved in 15mlwater. Next 30m1 glycerol is added
to the alginate
solution and mixed. Then 30g Surgihoney is then dissolved in the solution.
10m1 of Paraffin oil
is then added to the rheometer (TA Instruments AR-G2) which has a Jacket
Peltier and vane
geometry attached. 10m1 of Surgihoney solution is then added to the rheometer.
The rheometer
is then started under the following conditions: Shear rate 3000 us,
Temperature 40 C, gap
4000prn, Run time 10 minutes. After 1 minute 4m1 of PGPR (Polyglycerol
polyricinoleate) is
added. After 2 minutes 8m1 of non-aqueous calcium chloride solution (1M
Calcium chloride in
ethanol) is added dropwise to the rheometer. Once a total of 10 minutes has
elapsed, the
emulsion is transferred from the rheometer to a container.
Summary of emulsion formulations in Examples 7-11:
Table 10
fErnuIsioWSH (g) Glycerol Paraffin PGPR I NaAlg(g)/ Shear Temp. Order of
addition
cream (m1) Oil (ml) (ml) ' CaCl2(m1) rate
( C)
(1/s)
Ex 7 ' ''"Ter-- - 2000 37.5 Oil, then
PGPR,
then SH/glycerol
Ex 9 10 10 , 4, 6, 8, 4 3000 : 40.0
Oil and
or 10 SH/glycerol,
then
PGPR
........... 0.= ................
Ex 10 1.5 1.5 10 1 1/8-(-Wci) 2000 37.5 ..
Oil and PGPR;
then
SH/glycerol/NaAlg,
then CaCl2
Ex 11 30 30 ¨ 1/8 (non- 3000 40 Oil and
aq)
SH/glycerol/NaAlg(
aq), then PGPR,
then CaCl2 (non-
aq)
